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).