(24g) Altering Degradation Pathways Improves Protein Secretion in Cells Experiencing ER Stress

Therapeutic proteins from insulin to COVID-19 antibodies have reshaped modern health care. The ability to replace deficient proteins or to target problematic proteins serves as the treatment strategy for numerous diseases. The application of therapeutic proteins is limited by costs associated with manufacturing, purification, storage, and administration. Many therapeutic proteins must be made by mammalian cells to ensure biocompatible post-translational modifications, further increasing costs relative to production by microbial systems.

Protein overexpression can induce endoplasmic reticulum (ER) stress in mammalian cells and activate the unfolded protein response (UPR). The UPR decreases global protein production, increases chaperone production to refold misfolded proteins, increases protein degradation pathways, and, if necessary, initiates apoptosis. Activation of the UPR is unavoidable in therapeutic protein production, but a comprehensive understanding of the effects of the UPR on cell function would allow for mitigation of the negative effects of the UPR on protein production.

Minimizing ER stress can be achieved through proper management/selection of media compositions, agitation techniques, non-product protein expression, host cell line, and product gene sequence. An area that is understudied is the impact of UPR on the secretion of protein products. The UPR activates autophagy and proteasomal degradation. In autophagy stress causing components initiate a membrane formation event that forms a phagophore – a vesicle that engulfs the stress causing compounds. This phagophore matures into an autophagosome that eventually merges with acidic lysosomes for degradation of the contents. Rigid autophagosomal membranes protect the cell and neighboring cells from harm from the misfolded proteins by limiting their access to the cytoplasm and their secretion. In proteasomal degradation, ubiquitinated proteins must cross the ER membrane to enter the cytoplasm where proteasomes degrade misfolded proteins. Proteasomal degradation quickly returns amino acids to the cytoplasm for incorporation into newly synthesized proteins. During ER stress events, the activities of one or both degradation pathways is increased. As such, the levels of misfolded proteins secreted are reduced.

After translation, secreted proteins are inserted into the ER lumen where they are post –translationally modified before being packaged into COPII coated vesicles destined for the Golgi apparatus. At the Golgi, further post translational modifications are made, and proteins are packaged into secretory vesicles that are trafficked to the plasma membrane to release their cargo. Secretion is impeded by ER stress in several ways. Transcription of proteins is reduced by ER stress-induced epigenetic changes and mRNA translation is reduced by ER stress altering ribosome function. Autophagosomes form from ER membrane material and can sequester misfolded proteins that would otherwise be secreted. Finally, proteins exist along the secretory pathway that can transport ubiquitinated proteins across vesicle membranes for degradation by proteasomes. Understanding how to optimize the interaction between secretion and degradation of therapeutic proteins by cells experiencing ER stress is of importance to decreasing the costs of production.

In this work, we demonstrate that ER stress decreases therapeutic protein secretion through autophagosomal sequestration of the protein product and that increasing proteasomal degradation improves secretion. To isolate the impact of ER stress on secretion and not nuclear regulation, we used both pDNA and mRNA for secreted luciferase as model systems. ER stress was initiated by the n-glycosylation inhibitor tunicamycin (TM) with HeLa cells serving as the cell model. Increasing levels of TM decreased secretion efficiency - the amount of secreted luciferase in culture media compared to luciferase in cell lysates. The autophagy inhibitor 3-methyladenine improves secretion efficiency in ER-stressed cells with little impact on non-stressed cells, indicating autophagy as the cause of intracellular accumulation of secreted proteins. Finally, proteasome activation by Rolipram restore secretion efficiency in ER-stressed cells. Taken together our results inform biomanufacturing process design by indicating that increasing proteasomal degradation while reducing autophagy will improve the amount of secreted recombinant protein in culture media.