Inhibition of the ER-associated protein degradation mediator, VCP/p97, regulates organelle interaction

To mitigate ER stress, three major protein quality control systems are initiated to maintain proteostasis: the unfolded protein response (UPR), ER-associated protein degradation (ERAD), and autophagy. During ERAD, misfolded proteins must be exported from the ER to the cytosol by a dislocon complex. This process is energy-dependent, and VCP/p97, a multifunctional transitional endoplasmic reticulum ATPase (TER ATPase) acts as a protein unfolding machine with hundreds of substrates in diverse cellular pathways. VCP/p97 is critical for the regulation of proteostasis and the degradation of signaling related proteins, and is implicated in several diseases. Moreover, VCP is essential for maturation of ubiquitin-containing autophagosomes. Here, we show that VCP inhibition at low doses (~1 μM) has cytostatic effects in human umbilical vein endothelial cells (HUVECs) and that autophagy, a self-degradation process, plays a critical role as a survival mechanism. Furthermore, we identified an increase in cytosolic Ca2+ as a key trigger for autophagy, and propose Ca2+ translocation between ER-plasma membrane and ER-mitochondria (Mitochondria-associated ER membranes, MAMs). Interestingly, we observed differences in cell proliferation inhibition between VCP inhibitor treatment and siRNA-mediated VCP knockdown, suggesting multiple-targets of the VCP inhibitor. To identify these new potential binding targets of the VCP inhibitor, we performed a label-free target protein identification method, cellular thermal shift assay (CETSA)-LC-MS/MS analysis. In this presentation, we will focus on elucidating the novel mechanisms by which the VCP inhibitor and its target proteins regulate autophagy and organelle interaction.