Improving protein secretion with autophagy‐inhibiting or proteasome‐activating small molecules

Abstract BACKGROUND Therapeutic proteins are produced frequently 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, which can lead to endoplasmic reticulum (ER) stress and loss of proteostasis. In response, cells activate 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 impacts 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 (as a model for therapeutic protein production) and exposed to tunicamycin (TM) (an N‐glycosylation inhibitor that induces ER stress and the UPR). RESULTS As expected, TM exposure decreased luciferase production and secretion. Inhibiting autophagy improved luciferase secretion in stressed cells as expected. However, increasing proteasomal degradation also improved luciferase secretion while inhibiting proteasomal activity decreased secretion; thus, proteasomal activity was directly correlated to luciferase secretion. CONCLUSIONS Taken together, our results demonstrate that protein secretion can be improved through control of autophagy and proteasomal activity, providing insight into strategies for improving yield from protein production bioprocesses. © 2026 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).