The Pertussis Toxin S1 Subunit Is a Thermally Unstable Protein Susceptible to Degradation by the 20S Proteasome

Pande, Abhay H.; Moe, David; Jamnadas, Maneesha; Tatulian, Suren A.; Teter, Ken

doi:10.1021/bi061175+

Cited by 40 publications

(71 citation statements)

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“…It may also be susceptible to ubiquitin-independent degradation by the core 20S proteasome. [18][19]21 This general process has now been observed for three distinct ERtranslocating toxins. As such, A chain instability may represent a common strategy for triggering ERAD-mediated toxin translocation.…”

Section: Thermal Instability: a Common Theme For Toxin-erad Interactionsmentioning

confidence: 63%

“…19,20 Interestingly, though, both ricin A chain and PT S1 are degraded in vivo and/or in vitro by ubiquitin-independent proteasomal mechanisms. [18][19]21 These observations collectively suggest that A chain instability could represent a common theme in toxin-ERAD interactions.…”

Section: Introductionmentioning

confidence: 84%

“…[17][18]22,[57][58] Each toxin A chain is also stabilized by an association with its cytosolic target or other host factor. 17-18 Thus, A chain instability is only apparent after separation from the holotoxin in the ER and before interaction with a specific component of the host cell cytosol.…”

Section: Thermal Instability: a Common Theme For Toxin-erad Interactionsmentioning

confidence: 99%

“…Ricin A chain is refolded by an interaction with its ribosome target, while PT S1 is stabilized by the association with its NAD co-factor. 17,18 These events could allow the heat-labile A chains to assume active conformations in the cytosol and to avoid the usual proteolytic fate of ERAD substrates. Ricin A chain and PT S1 are further protected from ubiquitin-dependent proteasomal degradation because they have few (ricin A chain) or no (PT S1) lysine residues for ubiquitin conjugation.…”

Section: Introductionmentioning

confidence: 99%

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Conformational Instability of the Cholera Toxin A1 Polypeptide

Pande

Scaglione

Taylor

et al. 2007

Journal of Molecular Biology

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176

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SummaryCholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by vesicular transport. In the ER, the catalytic CTA1 subunit dissociates from the holotoxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). It is hypothesized that CTA1 triggers its ERAD-mediated translocation into the cytosol by masquerading as a misfolded protein, but the process by which CTA1 activates the ERAD system remains unknown. Here, we directly assess the thermal stability of the isolated CTA1 polypeptide by biophysical and biochemical methods and correlate its temperature-dependent conformational state with susceptibility to degradation by the 20S proteasome. Measurements with circular dichroism and fluorescence spectroscopy demonstrated that CTA1 is a thermally unstable protein with a disordered tertiary structure and a disturbed secondary structure at 37°C. A protease sensitivity assay likewise detected the temperature-induced loss of native CTA1 structure. This protease-sensitive conformation was not apparent when CTA1 remained covalently associated with the CTA2 subunit. Thermal instability in the dissociated CTA1 polypeptide could thus allow it to appear as a misfolded protein for ERADmediated export to the cytosol. In vitro, the disturbed conformation of CTA1 at 37°C rendered it susceptible to ubiquitin-independent degradation by the core 20S proteasome. In vivo, CTA1 was also susceptible to degradation by a ubiquitin-independent proteasomal mechanism. ADPribosylation factor 6, a cytosolic eukaryotic protein that enhances the enzymatic activity of CTA1, stabilized the heat-labile conformation of CTA1 and protected it from in vitro degradation by the 20S proteasome. Thermal instability in the reduced CTA1 polypeptide has not been reported before, yet both the translocation and degradation of CTA1 may depend upon this physical property.

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Section: Thermal Instability: a Common Theme For Toxin-erad Interactionsmentioning

confidence: 63%

Section: Introductionmentioning

confidence: 84%

Section: Thermal Instability: a Common Theme For Toxin-erad Interactionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Conformational Instability of the Cholera Toxin A1 Polypeptide

Pande

Scaglione

Taylor

et al. 2007

Journal of Molecular Biology

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176

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“…Reduction of the intramolecular PTS1 disulfide bond by ER-localized oxidoreductases could further destabilize the holotoxin and thereby assist PTS1 release from the PTB oligomer (25). Disassembly of the holotoxin leads to the spontaneous unfolding of PTS1, a thermally unstable protein (26) that is otherwise held in a stable conformation by its association with the PTB oligomer (27). This ER-localized unfolding event would likely identify PTS1 as a substrate for ERAD-mediated translocation to the cytosol.…”

mentioning

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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IDPs and Protein Degradation in the Cell

Shaul

Tsvetkov

Reuven

2010

Instrumental Analysis of Intrinsically Disordered Proteins

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1The degradation of the majority of cellular proteins is mediated by the proteasomes. Ubiquitin -dependent proteasomal protein degradation is executed by a number of enzymes that interact to modify the substrates prior to their engagement with the 26S proteasomes. The 26S proteasome is made of two complexes, the 20S and the 19S. The role of 19S is to unfold the proteins to gain entry into the 20S particle, where the protein is cleaved into short peptides. Thus, some of the functions of the 19S complex are expected to be dispensable for degradation of intrinsically disordered proteins, or IDPs. Indeed, in cell -free systems, at least some of the IDPs are digested by 20S particles in the absence of the 19S. In fact, it appears that susceptibility to the 20S proteasome may represent a hallmark of IDPs. Recent evidence suggests that IDPs are susceptible to degradation in vivo by the 20S proteasome as well. The process is ubiquitin independent and takes place by default. However, the process of default degradation can be regulated by different strategies. The described process of IDP degradation provokes new predictions and explanations in the fi eld of protein regulation and functionality. ABSTRACT

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The Pertussis Toxin S1 Subunit Is a Thermally Unstable Protein Susceptible to Degradation by the 20S Proteasome

Cited by 40 publications

References 50 publications

Conformational Instability of the Cholera Toxin A1 Polypeptide

Conformational Instability of the Cholera Toxin A1 Polypeptide

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

IDPs and Protein Degradation in the Cell

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