The autophagy-lysosomal pathway participates in protein QC by helping to remove protein aggregates. Protein aggregates or defective organelles are first segregated by an isolated double membrane (phagophore) to form an autophagosome which later fuses with lysosomes to form an autophagolysosome where the segregated content is degraded by lysosomal hydrolases (Levine & Kroemer, 2008). This type of autophagy is known as macroautophagy.
Macroautophagy can degrade all forms of misfolded proteins whereas proteasome degradation is likely limited to soluble proteins (Ding & Yin, 2008). Macroautophagy may be activated by UPS malfunction and/or aberrant protein aggregation to help remove aggregates (Rubinsztein, 2006). For example a toxic gain-of-function point mutation in alpha1-antitrypsin Z impairs correct protein folding, rendering the protein prone to form aggregated polymers within the hepatocyte ER. WT protein is primarily degraded by the proteasome, but the mutant
144
is thought to be degraded primarily by macroautophagy (Yorimitsu & Klionsky, 2007).
5.2 Cycloheximide chase and proteasome inhibition in WT stable
line cells
Many misfolded proteins are unstable within the cell and are rapidly degraded (Waters, 2001). One method by which protein stability can be assessed is by using cycloheximide and performing a cycloheximide chase experiment. Cycloheximide is produced by S. griseus and inhibits protein translation by inhibiting peptidyl transferase activity of the 60S ribosomal subunit (Schneider-Poetsch et al, 2010). By using cycloheximide to inhibit protein synthesis, the fate of the protein synthesised before inhibition can be analysed.
In order to investigate whether the mutants were rapidly degraded within the cell cycloheximide chase experiments were performed. In preliminary experiments cycloheximide (Sigma, dissolved in DMSO according to the manufacturer’s instructions) was first added to confluent stable line cells seeded in six well plates, expressing WT ADAMTS13 at various concentrations (ranging from 0 to 100µg/ml). The concentrations chosen were based upon those previously shown to be optimal for inhibition of protein synthesis (Gorner et al, 2004;Canaff et al, 2012;Park et al, 2012). After 24 hours cell lysates were harvested. This preliminary experiment was carried out to confirm that cycloheximide did inhibit protein synthesis at the concentrations chosen and also that cycloheximide was not toxic at these concentrations. The cells that were incubated with 100µg/ml cycloheximide appeared a lot smaller and ‘less healthy’ compared to at the other concentrations. Consequently this concentration was omitted in subsequent experiments.
Cell lysate samples are shown in Figure 5-3. The quantity of ADAMTS13 within the cell decreased in the presence of cycloheximide in a dose dependent manner, suggesting that cycloheximide was indeed preventing protein synthesis within the cell at the concentrations chosen.
145
Figure 5-3 Cycloheximide reduces protein synthesis in cells stably expressing WT ADAMTS13
Cell lysates are shown of cells stably expressing WT ADAMTS13 incubated with different concentrations of cycloheximide for 24 hours: 100µg/ml, 50µg/ml, 20µg/ml, 5µg/ml, and 1µg/ml and as a control no cycloheximide. Expected size of ADAMTS13 is ~190kDa. Results are from one experiment.
In order to investigate whether the mutants were degraded by the cell proteasome, the proteasome was inhibited using MG132, a peptide aldehyde that blocks the proteolytic activity of the 26S proteasome complex (Lee & Goldberg, 1998). This inhibitor was initially added to cells stably expressing WT ADAMTS13 to understand the effect of this inhibitor on the intracellular levels of WT ADAMTS13 and also how toxic this inhibitor was to the cells. WT ADAMTS13 stable line cells seeded in six well plates were incubated with various concentrations of MG132 varying from 0 to 100µM. The concentrations chosen were based upon those previously shown to be optimal for inhibition of the proteasome (Gorner et al, 2004;Canaff et al, 2012;Park et al, 2012;Jensen et al, 1995). Cell lysate samples were harvested 24 hours after incubation with MG132. Examination by light microscopy did not reveal any indication of extreme toxicity at these concentrations (i.e. there was no excessive cell death). However the cells were a lot smaller compared to negative control cells which had not been incubated with MG132. Cell lysate samples are shown in Figure 5-4, lanes 1-6. Although the lanes are heavily loaded, MG132 did not appear to cause a large increase or decrease in ADAMTS13 levels within the cell.
146
Figure 5-4 Proteasome inhibition in cells stably expressing WT ADAMTS13
Cell lysates of cells stably expressing WT ADAMTS13 after 24 hours incubation with various concentrations of MG132: 100µM, 50µM, 25µM, 10µM, 5µM, 1µM or no MG132 and incubated with or without 20µg/ml cycloheximide. Expected size of ADAMTS13 is ~190kDa. Results are from one experiment.
Stable line cells expressing WT ADAMTS13 seeded in six well plates were also incubated with these various concentrations of MG132 in the presence of 20µg/ml cycloheximide to understand if extreme toxicity occurred to the cells in the presence of both of these drugs. Cell lysate samples were harvested after 24 hours and no excessive cell death was observed. Cell lysate samples are shown in Figure 5-4, lanes 7-12.
The quantity of ADAMTS13 within the cell at the various different concentrations of MG132 was similar (Figure 5-4). Levels of ADAMTS13 in lanes 7-12 were lower compared to lanes 1-6 due to the additional presence of cycloheximide in the media of cells shown in lanes 7-12. Protein synthesis was reduced in these samples in agreement with the results from Figure 5-3. Together these results suggest that proteasome inhibition does not have a large effect on the intracellular levels of WT ADAMTS13.
147