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Title: The cytosolic fate of ricin A chain in target cells
Author: Hart, Philip John
ISNI:       0000 0004 2702 3210
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2010
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Ricin is a heterodimeric, toxic plant protein. It is able to deliver its catalytic A chain (RTA) into the cytosol of target cells. RTA crosses the endoplasmic reticulum (ER) membrane into the cytosol, masquerading as a substrate of ER-associated degradation (ERAD) to do so. Therein, RTA inactivates ribosomes. This thesis shows that RTA is prone to lose solubility in vitro near the physiological temperature and pH of target cells. This instability is hypothesised to cause RTA to misfold in the ER lumen, promoting chaperone and membrane interactions therein. Fittingly, this thesis shows that Grp94, a lumenal chaperone, promotes the toxicity of RTA, and that liposomes constructed of negatively-charged phospholipid interact with RTA in vitro. This instability resembles that of other toxic A chains that exploit ERAD. Cholera toxin A chain and pertussis toxin A chain, for instance, are also relatively unstable (Pande et al., 2007 & 2008). The Hrd1 complex now seems the strongest candidate for retrotranslocating RTA from the ER lumen (Li et al., 2010). In the cytosol, the proteasomal cap has been shown to be involved in downstream processing of RTA – enabling toxicity (Li et al., 2010). This thesis reports that, in mammals, the balance of cytosolic chaperones and their co-factors helps to dictate the success of retrotranslocated RTA, putatively by determining its escape from terminal degradation in the proteasomal core. The effect of these chaperones occurs at a stage beyond access of the toxin subunit to the ER, and can result in both activation and inactivation of cytosolic RTA. It has been shown that, on one hand, Hsc70 is responsible for activating RTA. Hsc70 may aid RTA in attaining an active conformation in the cytosol after retrotranslocation. Alternatively, it might supplant the effectors of its degradation. On the other hand, Hsc70 also enters RTA into a sequential triage with Hsp90. Unlike Hsc70, Hsp90 deactivates RTA. This effect is dependent upon the lysines of this toxin subunit, suggesting Hsp90 may participate in the lysine-ubiquitination of RTA. Supporting this conclusion, Hsc70 and Hsp90 can both ubiquitinate RTA in vitro. This ubiquitination can be promoted if RTA is first incubated with liposomes. This implies that RTA may be particularly vulnerable to ubiquitination during retrotranslocation, where it might also be partially solvated by phospholipid. Contrasting to RTA, Hsp90 actually aids the toxicity and dislocation of ER-retrotranslocating cholera toxin A chain (Taylor et al., 2010). It seems that RTA may have fortuitously evolved to exploit Hsc70 rather than Hsp90 to promote its cytosolic activation. Provocatively, the hydrophobic C-terminal tail of RTA demarcates it from a homologous toxin, saporin, which does not exploit ERAD to achieve toxicity. Indeed, this region may be an adaptation RTA has acquired to promote interaction of the toxin subunit with Hsc70, which even seems to occur in RTA’s native, folded state. This interaction may be another reason why the region is apparently significant to the cytotoxicity of the protein (Simpson et al., 1995) Finally, because the reactivation of RTA after retrotranslocation involves proteins with broad specificity (Hsc70, Hsp90), this thesis hypothesises that this pathway may operate for other ERAD substrates. Prior investigators have shown isolated examples of this phenomenon. For example the degradation-independent retrotranslocation of extracellularly-applied luciferase (Giodini & Cresswell, 2008) and of endogenous calreticulin (Afshar et al., 2006). This thesis hypothesises that the success of a protein in being reactivated post-dislocation will be determined by stringency of the cell’s chaperone network, the propensity of the substrate to be degraded, and its propensity to refold. As a protein which is toxic to the cell when refolded in the cytosol, RTA will be a useful tool to investigate this putative, broadly relevant, post-dislocation activation pathway.
Supervisor: Not available Sponsor: Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology