Tuhina Banerjee

Stabilization of the Tertiary Structure of the Cholera Toxin A1 Subunit Inhibits Toxin Dislocation and Cellular Intoxication

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic subunit of CT (CTA1) then crosses the ER membrane and enters the cytosol in a process that involves the quality control mechanism of ER-associated degradation. The molecular details of this dislocation event have not been fully characterized. Here, we report that thermal instability in the CTA1 subunit-specifically, the loss of CTA1 tertiary structure at 37 degrees C-triggers toxin dislocation. Biophysical studies found that glycerol preferentially stabilized the tertiary structure of CTA1 without having any noticeable effect on the thermal stability of its secondary structure. The thermal disordering of CTA1 tertiary structure normally preceded the perturbation of its secondary structure, but in the presence of 10% glycerol the temperature-induced loss of CTA1 tertiary structure occurred at higher temperatures in tandem with the loss of CTA1 secondary structure. The glycerol-induced stabilization of CTA1 tertiary structure blocked CTA1 dislocation from the ER and instead promoted CTA1 secretion into the extracellular medium. This, in turn, inhibited CT intoxication. Glycerol treatment also inhibited the in vitro degradation of CTA1 by the core 20S proteasome. Collectively, these findings indicate that toxin thermal instability plays a key role in the intoxication process. They also suggest the stabilization of CTA1 tertiary structure is a potential goal for novel antitoxin therapeutic agents.

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Protein-disulfide isomerase (PDI) has been proposed to exhibit an “unfoldase” activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin.

A Therapeutic Chemical Chaperone Inhibits Cholera Intoxication and Unfolding/Translocation of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels as an intact AB5 protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera.

Contribution of Subdomain Structure to the Thermal Stability of the Cholera Toxin A1 Subunit

The catalytic A1 subunit of cholera toxin (CTA1) is an ADP-ribosyltransferase with three distinct subdomains: CTA1(1) forms the catalytic core of the toxin, CTA1(2) is an extended linker between CTA1(1) and CTA1(3), and CTA1(3) is a compact globular region. CTA1 crosses the endoplasmic reticulum (ER) membrane to enter the cytosol where it initiates a cytopathic effect. Toxin translocation involves ER-associated degradation (ERAD), a quality control system that exports misfolded proteins from the ER to the cytosol. At the physiological temperature of 37 °C, the free CTA1 subunit is in a partially unfolded conformation that triggers its ERAD-mediated translocation to the cytosol. Thus, the temperature sensitivity of CTA1 structure is an important determinant of its function. Here, we examined the contribution of CTA1 subdomain structure to the thermal unfolding of CTA1. Biophysical measurements demonstrated that the CTA1(1) subdomain is thermally unstable and that the CTA1(2) subdomain provides a degree of conformational stability to CTA1(1). The CTA1(3) subdomain does not affect the overall stability of CTA1, but the thermal unfolding of CTA1 appears to begin with a local loss of structure in the CTA1(3) subdomain: glycerol and acidic pH both inhibited the thermal disordering of full-length CTA1 but not the disordering of a CTA1 construct lacking the A1(3) subdomain. These observations provide mechanistic insight regarding the thermal unfolding of CTA1, an event which facilitates its subsequent translocation to the cytosol.

Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit

Background: Cholera toxin enters the target cell in a disordered state and must attain a folded, active conformation to modify its G protein target. Results: Lipid rafts, where G protein is located, shift the disordered toxin to a functional conformation. Conclusion: Lipids rafts provide a chaperone-like function for cholera toxin. Significance: Lipid rafts play an important role in regulating toxin function through chaperone-like activity. Cholera toxin (CT) travels from the cell surface to the endoplasmic reticulum (ER) as an AB holotoxin. ER-specific conditions then promote the dissociation of the catalytic CTA1 subunit from the rest of the toxin. CTA1 is held in a stable conformation by its assembly in the CT holotoxin, but the dissociated CTA1 subunit is an unstable protein that spontaneously assumes a disordered state at physiological temperature. This unfolding event triggers the ER-to-cytosol translocation of CTA1 through the quality control mechanism of ER-associated degradation. The translocated pool of CTA1 must regain a folded, active structure to modify its G protein target which is located in lipid rafts at the cytoplasmic face of the plasma membrane. Here, we report that lipid rafts place disordered CTA1 in a functional conformation. The hydrophobic C-terminal domain of CTA1 is essential for binding to the plasma membrane and lipid rafts. These interactions inhibit the temperature-induced unfolding of CTA1. Moreover, lipid rafts could promote a gain of structure in the disordered, 37 °C conformation of CTA1. This gain of structure corresponded to a gain of function: whereas CTA1 by itself exhibited minimal in vitro activity at 37 °C, exposure to lipid rafts resulted in substantial toxin activity at 37 °C. In vivo, the disruption of lipid rafts with filipin substantially reduced the activity of cytosolic CTA1. Lipid rafts thus exhibit a chaperone-like function that returns disordered CTA1 to an active state and is required for the optimal in vivo activity of CTA1.

Substrate-induced unfolding of protein disulfide isomerase displaces the cholera toxin A1 subunit from its holotoxin.

To generate a cytopathic effect, the catalytic A1 subunit of cholera toxin (CT) must be separated from the rest of the toxin. Protein disulfide isomerase (PDI) is thought to mediate CT disassembly by acting as a redox-driven chaperone that actively unfolds the CTA1 subunit. Here, we show that PDI itself unfolds upon contact with CTA1. The substrate-induced unfolding of PDI provides a novel molecular mechanism for holotoxin disassembly: we postulate the expanded hydrodynamic radius of unfolded PDI acts as a wedge to dislodge reduced CTA1 from its holotoxin. The oxidoreductase activity of PDI was not required for CT disassembly, but CTA1 displacement did not occur when PDI was locked in a folded conformation or when its substrate-induced unfolding was blocked due to the loss of chaperone function. Two other oxidoreductases (ERp57 and ERp72) did not unfold in the presence of CTA1 and did not displace reduced CTA1 from its holotoxin. Our data establish a new functional property of PDI that may be linked to its role as a chaperone that prevents protein aggregation.

ADP‐ribosylation factor 6 acts as an allosteric activator for the folded but not disordered cholera toxin A1 polypeptide

The catalytic A1 subunit of cholera toxin (CTA1) has a disordered structure at 37°C. An interaction with host factors must therefore place CTA1 in a folded conformation for the modification of its Gsα target which resides in a lipid raft environment. Host ADP‐ribosylation factors (ARFs) act as in vitro allosteric activators of CTA1, but the molecular events of this process are not fully characterized. Isotope‐edited Fourier transform infrared spectroscopy monitored ARF6‐induced structural changes to CTA1, which were correlated to changes in CTA1 activity. We found ARF6 prevents the thermal disordering of structured CTA1 and stimulates the activity of stabilized CTA1 over a range of temperatures. Yet ARF6 alone did not promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted disordered CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6. Thus, ARF alone is unable to activate disordered CTA1 at physiological temperature: additional host factors such as lipid rafts place CTA1 in the folded conformation required for its ARF‐mediated activation. Interaction with ARF is required for in vivo toxin activity, as enzymatically active CTA1 mutants that cannot be further stimulated by ARF6 fail to intoxicate cultured cells.

Co- and Post-translocation Roles for HSP90 in Cholera Intoxication

Background: The unfolded A1 subunit of cholera toxin (CT) enters the host cytosol by passing through a pore in the endoplasmic reticulum (ER) membrane. Results: ATP-dependent refolding of CTA1 by Hsp90 is sufficient for toxin export to the cytosol. Conclusion: Hsp90 couples CTA1 refolding with CTA1 extraction from the ER. Significance: This work provides a molecular basis for toxin translocation into the host cytosol. Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the rest of the toxin. CTA1 then unfolds and passes through an ER translocon pore to reach its cytosolic target. Due to its intrinsic instability, cytosolic CTA1 must be refolded to achieve an active conformation. The cytosolic chaperone Hsp90 is involved with the ER to cytosol export of CTA1, but the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Moreover, potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 have not been explored. Here, we show by isotope-edited Fourier transform infrared 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 refolding of CTA1 by Hsp90 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance 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 allows disordered CTA1 to attain an active state, which is further enhanced by ADP-ribosylation factor 6, a host cofactor for CTA1. Our data indicate CTA1 translocation involves a process that couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. The molecular basis for toxin translocation elucidated in this study may also apply to several ADP-ribosylating toxins that move from the endosomes to the cytosol in an Hsp90-dependent process.

Identification of Molecular-Mimicry-Based Ligands for Cholera Diagnostics using Magnetic Relaxation

When covalently bound to an appropriate ligand, iron oxide nanoparticles can bind to a specific target of interest. This interaction can be detected through changes in the solutions spin-spin relaxation times (T2) via magnetic relaxation measurements. In this report, a strategy of molecular mimicry was used in order to identify targeting ligands that bind to the cholera toxin B subunit (CTB). The cellular CTB-receptor, ganglioside GM1, contains a pentasaccharide moiety consisting in part of galactose and glucose units. We therefore predicted that CTB would recognize carbohydrate-conjugated iron oxide nanoparticles as GM1 mimics, thus producing a detectable change in the T2 relaxation times. Magnetic relaxation experiments demonstrated that CTB interacted with the galactose-conjugated nanoparticles. This interaction was confirmed via surface plasmon resonance studies using either the free or nanoparticle-conjugated galactose molecule. The galactose-conjugated nanoparticles were then used as CTB sensors achieving a detection limit of 40 pM. Via magnetic relaxation studies, we found that CTB also interacted with dextran-coated nanoparticles, and surface plasmon resonance studies also confirmed this interaction. Additional experiments demonstrated that the dextran-coated nanoparticle can also be used as CTB sensors and that dextran can prevent the internalization of CTB into GM1-expressing cells. Our work indicates that magnetic nanoparticle conjugates and magnetic relaxation detection can be used as a simple and fast method to identify targeting ligands via molecular mimicry. Furthermore, our results show that the dextran-coated nanoparticles represent a low-cost approach for CTB detection.

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit(1). These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol(2-4). In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target(5). The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER(6-12). To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)(13-15). The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.