Lipid-Modulated,\nGraduated Inhibition of N‑Glycosylation\nPathway Priming Suggests Wide Tolerance of ER Proteostasis to Stress

Protein N-glycosylation is a cotranslational modification\nthat\ntakes place in the endoplasmic reticulum (ER). Disruption of this\nprocess can result in accumulation of misfolded proteins, known as\nER stress. In response, the unfolded protein response (UPR) restores\nproteostasis or responds by controlling cellular fate, including increased\nexpression of activating transcription factor 4 (ATF4) that can lead\nto apoptosis. The ability to control and manipulate such a stress\npathway could find use in relevant therapeutic areas, such as in treating\ncancerous states in which the native ER stress response is often already\nperturbed. The first committed step in the N-glycosylation pathway\nis therefore a target for potential ER stress modulation. Here, using\nstructure-based design, the scaffold of the natural product tunicamycin\nallows construction of a panel capable of graduated inhibition of\nDPAGT1 through lipid-substituent-modulated interaction. The development\nof a quantitative, high-content, cellular immunofluorescence assay\nallowed precise determination of downstream mechanistic consequences\n(through the nuclear localization of key proxy transcription factor\nATF4 as a readout of resulting ER stress). Only the most potent inhibition\nof DPAGT1 generates an ER stress response. This suggests that even\nlow-level “background” biosynthetic flux toward protein\nglycosylation is sufficient to prevent response to ER stress. “Tuned”\ninhibitors of DPAGT1 also now seemingly successfully decouple protein\nglycosylation from apoptotic response to ER stress, thereby potentially\nallowing access to cellular states that operate at the extremes of\nnormal ER stress.