Rheb[T23M] and [E40K] drive increased protein synthesis and secretion dependent on ER stress
To test whether Rheb mutants drive an increase in recombinant protein production in CHO cells, we transfected FLuc-CHO cells with vectors encoding Rheb mutants and performed firefly luciferase assays every 48 h for 7 days. In order to determine if the protein production capacity ofindividual cells was increased (and to take into account possible differences in cell number due to the effect of Rheb mutants on cell proliferation), the firefly luciferase assays were normalised to the number of cells present as counted on a haemocytometer. Cells expressing Rheb[T23M] showed a small yet significant increase in Firefly luciferase (Fig. 4a/b) indicating an increase in protein production. Interestingly, other Rheb mutants did not significantly increase the accumulation of firefly luciferase suggesting that while they promote constitutive mTORC1 activity, this does not result in universal upregulation of protein production.
Next, we sought to determine the effect of Rheb mutants on the output of a secreted protein, which is the key parameter for the production of recombinant proteins of commercial interest, which are harvested from the culture medium. To do this, we transfected GLuc-CHO cells with vectors encoding Rheb mutants and performed Gaussia luciferase assays on the growth media. As with the firefly luciferase assays, results were normalised to cell number to give an indication of accumulated Gaussia luciferase secretion on a per-cell basis. In contrast to firefly luciferase production, Rheb[T23M], [Y35N] and [E40K] all promoted a significant increase in GLuc secretion as calculated in this way (Fig. 4c). Cells expressing these mutants showed an approximately 3-fold increase in GLuc secretion after 7 days; no difference was seen in amounts of intracellular GLuc (not shown), consistent with Rheb mutants boosting secretion of the additional GLuc. As expected, treatment with the potent mTOR inhibitor AZD8055 (Chresta et al., 2010) reversed the effect of mutant Rheb (Fig. 4c/d). These data suggest that, as well as increasing protein synthesis, Rheb mutants may also increase cells’ secretory capacity, consistent with the findings for HEK293 cells. As increased ER volume is known to increase secretory capacity (Budge et al., 2020; M. Wang & Kaufman, 2016), we also performed the GLuc assay using cells treated with a combination of the ER stress response inhibitors iPERK (Axten et al., 2012), a compound that inhibits the kinase activity of PERK, and ISRIB (Sidrauski et al., 2013), a compound that interferes with the ability of phosphorylated eIF2 to inhibit the guanine exchange of eIF2B (Sidrauski, McGeachy, Ingolia, & Walter, 2015). Inhibition of the ER stress response dramatically decreased the secretion of GLuc from cells expressing Rheb mutants (Fig. 4e) indicating Rheb mediated ER expansion is crucial for increased GLuc secretion. Consistent with this, western blot analysis of GLuc-CHO cells transiently expressing Rheb mutants or an empty vector show that Rheb[T23M] and [E40K] significantly drive increased expression of the ATF4 arm of the UPR (Fig. 4f).