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Experimental Design and the Importance of Controls

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Published: Tue, 08 May 2018

  • Emma Powell
  1. Were the conclusions you drew from your submitted manuscript valid? Explain your reasoning.

The conclusions are invalid as there is little supporting evidence and controls are absent. There are no controls within either experiment and so the variables have not been investigated. For example, in the first experiment, without controls, it is difficult to distinguish between the effects of the inhibitor and ricin on protein synthesis. Furthermore, in the second experiment, without a galactose control, it is impossible to determine whether yeast cells grow successfully on this carbon source.

Even when controls are implemented, research has shown that ricin is “a poor substrate for proteasomal degradation” (Pietroni, et al., 2013) and that an increase in ricin sensitivity “simply reflects toxicity of the inhibitors themselves” (Pietroni, et al., 2013).

The conclusion that proteasome up-regulation could be a potential therapeutic is invalid as this would be more likely to inflict toxic effects on the cell (by disrupting cell homeostasis). Also, applying the same therapy for Shiga and ricin poisoning would be ineffective. Although both Shiga and ricin toxins utilise endocytosis as a mechanism for entering the cell, the downstream proteins that they bind to are significantly diverse.

  1. What controls should have been performed to support (or negate) your conclusions?

Within the first experiment, a dimethyl sulphoxide (DMSO) control should have been included. DMSO may have possible interactions with clasto Lactacystin β-lactone (cLβ-1) and may enhance its potency as an inhibitor. Therefore, this could result within the IC50 of cLβ-1 being lower than would normally be expected. Also, it could be that DMSO itself may be interfering with protein synthesis.

Another control that was required was the independent testing of cLβ-1. It could be that cLβ-1 is itself exerting toxic effects on the cell (such as through its indirect effects of targeting other proteases and consequently deregulating cell homeostasis) and is therefore responsible for the increased reduction in protein synthesis. A different protease inhibitor such as pepstatin should be included. As this does not target the proteasome, then a wild-type response would be observed. A positive control of protein inhibition is also required (such as neomycin). Figure 1 illustrates a potential new plate layout.

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In the yeast expression study, the choice of carbon source rather than the toxic effects of the A chain (RTA) may have affected the growth of the yeast. Research “discovered that glucose withdrawal from the growth medium led to a rapid inhibition of protein synthesis” (Ashe, et al., 1999). The control could have included yeast cells that were transformed with a plasmid containing a galactose-inducible protein that was not toxic. The yeast could have been subsequently grown on a galactose medium. If there was little growth of yeast then this would suggest that galactose was a mediocre carbon source.

  1. How can the experimental design be improved?

The experiment on HeLa cells could be improved by completing a serial dilution of the cLβ-1 in DMSO. This would determine the effects of different concentrations of inhibitor on protein synthesis and would provide a more accurate IC50. The investigator could also conduct a cell count to ascertain if too high a concentration of inhibitor exerts toxic effects on the cell. From this, the investigator could identify the optimal concentration of inhibitor to be used within the assay in the presence of ricin. If this was to be undertaken, a serial dilution of DMSO in growth medium should also be performed. This would ensure that there was a DMSO control for each concentration of inhibitor.

The investigators only studied one time point within their experiment (ricin was incubated with HeLa cells for 6 hours). The investigators could perform a time series alongside a serial dilution to ascertain the time point and concentration in which ricin exerts the greatest toxic effect.

The experiment could also be repeated several times to ensure that the results collected are not simply down to chance. Statistical analysis, such as a two-tailed t-test could be conducted to ascertain whether there is a significant difference between the effects of ricin alone and treatment with ricin and cLβ-1 on protein synthesis. Error bars should be included within the new figure generated.

In the yeast expression study, the yeast could be grown within liquid culture so that a cell count could be conducted using a haemocytometer. The gene CUP1 (confers copper resistance and is specifically found within yeast) could be incorporated into the plasmid under the same galactose-inducible promoter that controls RTA (Koller, et al., 2000). This would ensure that both the RTA gene had been inserted into the plasmid and that transformation of yeast was successful. Little or no growth of yeast cells in the presence of copper would imply that yeast had not been transformed successfully.

  1. Design a new experiment or experiments to test a proposition related to the content already presented.

One experiment could investigate how some of the cytosolic RTA is able to avoid degradation by the proteasome. Research has suggested that several toxins, including ricin have a low number of lysine residues (Deeks, et al., 2002). Ubiquitination may possibly occur at these residues and therefore, if few lysine residues are present, this may decrease the probability of polyubiquitination. As a consequence, the toxin will not be targeted to the proteasome. The number of lysine residues could be increased (however without affecting RTA’s function and stability) to create a polyubiquitination tag and whether this subsequently targets the RTA to the proteasome. Both wild –type and mutant forms of RTA could be run on an SDS-PAGE; if the mutant RTA band is absent from the gel then this suggests that it is possibly degraded by the proteasome.

Recent research has suggested that “Hsc70 cochaperone activity determines the fate of dislocated RTA” (Spooner, et al., 2008). One experiment could include reducing expression of specific chaperones and their cochaperones to identify those that are either required for ricin refolding or its targeting to the proteasome.

A pulse chase experiment could be conducted within yeast, with RTA being radiolabelled to track its location and pathway throughout the cell. This can ascertain whether it interacts with the proteasome. Also, this enables the percentage of RTA that avoids the proteasome to be calculated. Mutagenesis of E3 ligase and the proteasome can be created to see if this affects the movement and location of RTA.

Bibliography

Ashe, M., Long, S. & Sachs, A., 1999. Glucose Depletion Rapidly Inhibits Translation Initiation in Yeast. Molecular Biology of the Cell, 11(3).

Deeks, E. et al., 2002. The Low Lysine Content of Ricin A Chain Reduces the Risk of Proteolytic Degradation after Translocation from the Endoplasmic Reticulum to the Cytosol. Biochemistry, 10(4), pp. 3405-3413.

Koller, A., Valesco, J. & Subramani, S., 2000. The CUP1 promoter of Saccharomyces cerevisiae is inducible by copper in Pichia pastoris. Yeast, pp. 651-656.

Pietroni, et al., 2013. The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain. Biochemical Journal, Volume 453, pp. 435-445.

Spooner, R. et al., 2008. Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum. PNAS, 105(45), p. 17408–17413.


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