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bioRxiv · 10.1101/2025.08.05.666879

Secreted Protein Production is Improved by Controlling Endoplasmic Reticulum Stress Associated Protein Degradation

Splichal, R. C.·Chan, C.·Walton, S. P.
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Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/666879v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@9139c3org.highwire.dtl.DTLVardef@673bc1org.highwire.dtl.DTLVardef@1842210org.highwire.dtl.DTLVardef@1d86903_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG 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 loss of proteostasis and endoplasmic reticulum (ER) stress. 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) (to activate ER stress). As expected, TM exposure decreased protein production and secretion. Inhibiting autophagy improved secretion in stressed cells as expected. However, counterintuitively, increasing proteasomal degradation improved secretion while inhibiting proteasomal activity decreased secretion, that is proteasomal activity was directly correlated to secretion. 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. Key PointsO_LITunicamycin induced ER stress reduced protein production. C_LIO_LIAutophagy inhibition improved secretion in ER stressed cells. C_LIO_LIActivation of proteasomal degradation improved secretion in ER stressed cells. C_LI

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Splichal, R. C., Chan, C., Walton, S. P.. 2025-08-07. Secreted Protein Production is Improved by Controlling Endoplasmic Reticulum Stress Associated Protein Degradation. https://doi.org/10.1101/2025.08.05.666879

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