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

Ray, Supriyo; Taylor, Michael; Banerjee, Tuhina; Tatulian, Suren A.; Teter, Ken

doi:10.1074/jbc.m112.385575

Cited by 29 publications

(31 citation statements)

References 66 publications

(69 reference statements)

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“…The G protein target of CTA1 is located in plasma membrane lipid rafts that have been shown to induce a gain-of-structure and function in disordered CTA1 (46). Previous experiments verified that lipid rafts did not dislodge Hsp90 from CTA1 (Table 2).…”

Section: Cta1 Activity Is Enhanced By Host Factors In a Sequence-depementioning

confidence: 72%

“…ARF6 was generated as previously described (49). Large unilamellar vesicles (LUVs) mimicking the composition of the plasma membrane or lipid rafts were generated as previously described (46).…”

Section: Methodsmentioning

confidence: 99%

“…Lipid rafts were recently shown to exhibit a "lipochaperone" property that places disordered CTA1 in a folded, functional state at physiological temperature. Furthermore, lipid rafts are essential for the in vivo activity of cytosolic CTA1 (46). Other host factors that could refold and/or activate cytosolic CTA1 have yet to be identified.…”

mentioning

confidence: 99%

“…Refolding will not occur spontaneously because CTA1 is an unstable protein. In fact, the isolated CTA1 polypeptide has little to no enzymatic activity at 37°C (45,46). An interaction with ARF proteins will enhance the activity of folded CTA1 and is required for productive intoxication of cultured cells, but ARF alone cannot induce a gain-of-structure or gain-of-function in disordered CTA1 (47).…”

mentioning

confidence: 99%

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

Burress

Taylor

Banerjee

et al. 2014

Journal of Biological Chemistry

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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.

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Section: Cta1 Activity Is Enhanced By Host Factors In a Sequence-depementioning

confidence: 72%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Burress

Taylor

Banerjee

et al. 2014

Journal of Biological Chemistry

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“…An interaction with NAD, the donor molecule for the ADP-ribosylation reaction, may stabilize the cytosolic pool of PTS1 (26). Other host factors may also stabilize and/or activate PTS1 in the cytosol, similar to the interactions between a variety of host factors and either the ricin A chain (RTA) or cholera toxin A1 chain (CTA1) (30)(31)(32)(33)(34)(35)(36). PTS1 moves from the ER to the cytosol, but the in vivo importance of PTS1 instability and a functional role for ERAD in PTS1 translocation have not been established.…”

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confidence: 99%

Thermal Unfolding of the Pertussis Toxin S1 Subunit Facilitates Toxin Translocation to the Cytosol by the Mechanism of Endoplasmic Reticulum-Associated Degradation

et al. 2016

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bPertussis toxin (PT) moves from the host cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic PTS1 subunit dissociates from the rest of the toxin in the ER and then shifts to a disordered conformation which may trigger its export to the cytosol through the quality control mechanism of ER-associated degradation (ERAD). Functional roles for toxin instability and ERAD in PTS1 translocation have not been established. We addressed these issues with the use of a surface plasmon resonance system to quantify the cytosolic pool of PTS1 from intoxicated cells. Only 3% of surface-associated PTS1 reached the host cytosol after 3 h of toxin exposure. This represented, on average, 38,000 molecules of cytosolic PTS1 per cell. Cells treated with a proteasome inhibitor contained larger quantities of cytosolic PTS1. Stabilization of the dissociated PTS1 subunit with chemical chaperones inhibited toxin export to the cytosol and blocked PT intoxication. ERAD-defective cell lines likewise exhibited reduced quantities of cytosolic PTS1 and PT resistance. These observations identify the unfolding of dissociated PTS1 as a trigger for its ERAD-mediated translocation to the cytosol. The AB toxins are composed of a catalytic A moiety and a cellbinding B moiety. Some AB toxins move by retrograde vesicular transport from the cell surface to the endosomes, from the endosomes to the Golgi apparatus, and from the Golgi apparatus to the endoplasmic reticulum (ER), where A/B subunit dissociation occurs (1). Unfolding of the dissociated A chain places the toxin in a translocation-competent conformation and activates the host mechanism of ER-associated degradation (ERAD) (2). This quality control system exports misfolded proteins from the ER to the cytosol through protein-conducting channels in the ER membrane (3). ERAD substrates are usually degraded by the cytosolic ubiquitinproteasome system, but the A chains of ER-translocating toxins effectively evade this pathway because they lack the lysine residues required for ubiquitin conjugation (4-7).Pertussis toxin (PT) is an AB 5 -type, ER-translocating toxin (8, 9). Its A subunit, PTS1, disrupts host signaling events through the ADP-ribosylation of G␣ proteins. The B subunit is a hetero-oligomer composed of four proteins (PTS2, PTS3, two copies of PTS4, and PTS5) that bind to specific but largely unidentified glycoconjugates on the host cell (10-14). Holotoxin assembly involves noncovalent positioning of PTS1 above and partially within the central pore of the ring-like PTB oligomer (15). As with other ER-translocating toxins, PT transport from the cell surface to the ER appears to be an inefficient process: fluorescence microscopy, immunoelectron microscopy, and subcellular fractionation can detect internalized toxin in the endosomes and Golgi apparatus but not the ER (16-19). The inhibition of PT intoxication with brefeldin A (BfA), a drug that blocks retrograde vesicular transport to the ER, strongly suggests the functional pool of toxin must move from ...

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Engineering the Composition of Heterogeneous Lipid Bilayers to Stabilize Tethered Enzymes

Black

Kienle

Kaar

et al. 2020

Adv Materials Inter

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As surfaces for enzyme immobilization, lipid bilayers (LBs) have the potential to exhibit chaperone‐like activity, which can greatly enhance enzyme stability. Here, it is shown that this stabilizing effect can be generalized to a broad range of enzymes by modifying the mixed lipid composition based on the properties of the enzyme. This is demonstrated by analyzing the stability of nitroreductase, lipase, organophosphorus hydrolase, and lysozyme on LBs composed by mixing zwitterionic 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine with anionic 1,2‐dioleoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol) or cationic 1,2‐dioleoyl‐sn‐glycero‐3‐ethylphosphocholine. Interestingly, while the optimal lipid composition exhibits a zeta potential with the same sign as each enzyme, the stabilizing compositional ranges are broad, and mixed lipid LBs are more stabilizing than homogeneous LBs with similar charge. This suggests that net charge and compositional heterogeneity are both important factors. Moreover, the degree of stabilization on appropriate bilayers under extremely denaturing conditions (7 m urea) is unprecedented, and activity of the enzymes is up to three‐fold greater than that of the soluble enzyme at each enzyme's temperature optimum. By correlating enzyme diffusion on LBs with information about re‐folding kinetics derived from single‐molecule measurements, the influence of charge and composition on enzyme‐LB interactions is shown, providing a rationale for the stabilizing effect of heterogeneous LBs.

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

Cited by 29 publications

References 66 publications

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

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

Thermal Unfolding of the Pertussis Toxin S1 Subunit Facilitates Toxin Translocation to the Cytosol by the Mechanism of Endoplasmic Reticulum-Associated Degradation

Engineering the Composition of Heterogeneous Lipid Bilayers to Stabilize Tethered Enzymes

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