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Pre-blocking of the incubation vessel - If these in vitro findings were to be extended, it

would be advisable to block binding to the incubation vessel beforehand by flushing it with low concentrations of a relatively inert protein (Van den Berg et al., 1999). Alternatively, a material which provides a polar rather than a hydrophobic surface could be chosen for the incubation vessel. This would increase the proportion observable in the soluble fraction under untreated conditions and would increase the dynamic range of the assay. This would enable material which has truly aggregated to be distinguished from material which has adhered to the reaction vessel. Without such blocking, the assay used in this chapter measures both of these phenomena.

The observation of this tendency of RTA to adhere to hydrophobic surfaces, however, is not without meaningful application. The results presented in this chapter have indicated that the C-terminus of RTA is likely to be responsible for this binding and may also interact with Hsc70 while the toxin subunit is in its native conformation. This may well result in an ongoing interaction with Hsc70 long after membrane retrotranslocation. As the C-terminus is functionally implicated in the toxicity of RTA (Simpson et al., 1995), judicious experiments using unblocked tubes might provide an experimental system useful in its investigation.

Screening for destabilising ER factors and stabilising cytosolic factors – If a systematic

way for producing the cytosolic extract could be established which also maintained long-term solubilising activity, it would be a useful tool. The extract could be fractionated and screened for proteins with solubilising or pro-aggregation activity. Fractionation could be repeated iteratively (with progressive refinement) until isolated protein species with significant effects were identified. These species could then be identified by mass spectrometry. By such a method, a whole host of other “pro-solubility” or “pro-aggregation” factors could be discovered which might be significant interacting partners of RTA in vivo.

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It might be expected that the toxin subunit has evolved to exploit the physiological conditions of the ER and cytosolic compartments in different ways, tailored to its toxic motives. Unfortunately, the complexity of these compartments is difficult to meaningfully disassemble using the solubility assay – especially for broadly important factors like calcium ion concentration (cf. section 4.5.2.1). Synergy between one aspect and another in the more complex system that undeniably exists in vivo will likely drastically alter otherwise simplistically-observed trends. For instance: the overlaid functionality of the protein matrix in response to these chemical factors. The concentration of calcium in the ER – as a particular example – modulates the ERAD of many secretory proteins (Van Anken & Braakman, 2005). So, too, do changes in redox levels and the pH (Sevier & Kaiser, 2008). To provide a more exhaustive study of any physiologically relevant chemical factors that might affect the conformational stability of RTA, however, would be intriguing. The most obvious candidates for future investigation would be the lipid environment and a comparison of the chaperone complements of both the ER and cytosol.

Screening for antagonism of the negatively-charged liposome interaction – The interaction

of RTA with liposomes constructed from exclusively POPS could not be countered simply with the use of a small molecular chaperone (as predicted from the hypotheses of Sandvig et al., 1984). However, glycerol might be able to compete against liposomes where POPS has been diluted with neutral lipid. Identification of other factors which could counter this interaction would be particularly interesting to study. This is because no such factor has yet been proposed to suggest how RTA would be prevented from interacting with the cytosolic leaflet of the ER membrane, just as it is proposed to in the ER (Mayerhofer et al., 2009). This problem is highlighted by reports that lipid distribution between the two leaflets of the ER membrane is highly symmetrical (Pomorski & Menon, 2006). This precludes the enrichment of negatively-charged phospholipids in the ER-leaflet being the mechanism. It is a possibility that chaperones like Hsc70 might be responsible for sustaining the cytosolic solubility of RTA and preventing such a regressive membrane interaction. Intriguingly, interactions with the membrane are thought to be caused by the C-terminus of RTA (Mayerhofer et al., 2009). That Hsc70 seems to bind this region at 37°C and prevent it from associating with a hydrophobic surface in vitro may be an analogous feat.

Alternatively, the solubility assay could be used to determine if RTA can be removed from a liposome-bound state after having already been allowed to interact. RTA and liposomes could be incubated together, and factors added to see if they could subsequently disengage

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such a complex. If the supply of Hsc70 had not been limiting, experiments like this would have been undertaken to test this possibility. Other factors that would have been interesting to study in this regard include the regulatory 19S proteasomal caps and p97-containing complexes, which have a similar role during ERAD (Meusser et al., 2005).

Identification of potential storage excipients - Incidentally, the methods demonstrated in this

chapter could also be used as a tool to study potential excipients with which RTA may be stored for long periods of time. Such a study is of special interest to those who wish to develop and store vaccines (Peek et al., 2006). Currently, our laboratory stores RTA in a pH6.4, sodium phosphate buffer – often containing KCl or glycerol, which stabilises the solubility of RTA. These conditions could be optimised, using this assay to maximise the chronic solubility of RTA. Peek et al. (2006) used a similar assay, where they studied the effect of candidate chemicals on the turbidity of a dilute solution of RTA over time. However, they discovered this assay was inappropriate in the case of chemicals which themselves contributed to turbidity. The techniques used herein would not be affected in this way and so might prove complementary.

Alternative assays – Other assays for determining the interaction of two proteins (such as Hsc70 with RTA) do exist. For instance: analytical ultra-centrifugation, gel filtration, fluorescence resonance energy transfer (FRET) and surface plasmon resonance. If the experimenter wished to support the data herein, those assays might prove a useful supplement to those used herein. For the interaction of factors such as salt, glycerol and pH, fluorescence could even be measured.