Frequently, therapeutic proteins are produced by mammalian cells in large-scale bioreactors. As a result, producer cells are exposed to a chemically (nutrients, gas exchange, target protein overexpression) and physically (shear due to mixing) stressful environment. In response, cells increase protein synthesis (e.g., of chaperones), which, along with overexpression of the product protein, overwhelms the quality control mechanisms in the endoplasmic reticulum (ER), activating the unfolded protein response (UPR). The UPR includes activation of autophagy and proteasomes, both of which target unfolded/misfolded proteins for degradation. To investigate the impact of autophagy and proteasome activity on secreted protein production in ER-stressed cells, we used HeLa and MDA-MB-231 cells transfected to express Gaussia luciferase and tunicamycin (TM) to activate ER stress. TM exposure decreased protein production and secretion. Inhibiting autophagy improved secretion in ER stressed cells. Inhibiting proteasome activity decreased secretion, and, conversely, activating proteasomes improved secretion. Taken together, these results showed that inhibition of autophagy and, somewhat counterintuitively, activation of proteasomal degradation improve protein secretion and production.
