Lipid-Modulated, Graduated Inhibition of N-Glycosylation Pathway Priming Suggests Wide Tolerance of ER Proteostasis to Stress

Protein N-glycosylation is a cotranslational modification that takes place in the endoplasmic reticulum (ER). Disruption of this process can result in accumulation of misfolded proteins, known as ER stress. In response, the unfolded protein response (UPR) restores proteostasis or responds by controlling cellular fate, including increased expression of activating transcription factor 4 (ATF4) that can lead to apoptosis. The ability to control and manipulate such a stress pathway could find use in relevant therapeutic areas, such as in treating cancerous states in which the native ER stress response is often already perturbed. The first committed step in the N-glycosylation pathway is therefore a target for potential ER stress modulation. Here, using structure-based design, the scaffold of the natural product tunicamycin allows construction of a panel capable of graduated inhibition of DPAGT1 through lipid-substituent-modulated interaction. The development of a quantitative, high-content, cellular immunofluorescence assay allowed precise determination of downstream mechanistic consequences (through the nuclear localization of key proxy transcription factor ATF4 as a readout of resulting ER stress). Only the most potent inhibition of DPAGT1 generates an ER stress response. This suggests that even low-level "background" biosynthetic flux toward protein glycosylation is sufficient to prevent response to ER stress. "Tuned" inhibitors of DPAGT1 also now seemingly successfully decouple protein glycosylation from apoptotic response to ER stress, thereby potentially allowing access to cellular states that operate at the extremes of normal ER stress.