Chemical and cell biological approaches to study ER stress-induced apoptosis.

摘要

Proper endoplasmic reticulum (ER) function is crucial to many aspects of cell physiology. Accordingly, cells react rapidly to correct ER dysfunction through a set of pathways known collectively as the ER stress response (ESR). However, when ER stress is persistent or strong, metazoan cells can induce apoptosis, or programmed cell death. ER stress-induced apoptosis has been implicated in many pathologies but remains poorly understood. In particular, new research tools are needed to study ER stress-induced apoptosis in mammals and to explore how this pathway might be manipulated for therapeutic benefit.; We have undertaken chemical and cell biological approaches to study mammalian ER stress-induced apoptosis. In a screen for small molecules that protect cells from ER stress-induced apoptosis (Chapter 2), we identified salubrinal (sal), a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2 subunit alpha (eIF2alpha). Sal also blocks eIF2alpha dephosphorylation mediated by a herpes simplex virus protein and inhibits viral replication. Thus, selective chemical inhibitors of eIF2alpha dephosphorylation may be useful in diseases involving ER stress or viral infection. More broadly, sal demonstrates the feasibility of selective pharmacological targeting of cellular dephosphorylation events.; We also used sal as a tool to discover new biology related to the ESR. In a search for cellular effects of sal (Chapter 3), we found that eukaryotic translation elongation factor 2 (EF-2) was phosphorylated in response to both sal treatment and ER stress itself. EF-2 phosphorylation in both cases requires EF-2 kinase (EF2K). EF2K-/- cells are resistant to ER stress-induced apoptosis, demonstrating a novel role for EF2K in apoptosic signaling downstream of the ESR.; Finally, we used chemical and genetic approaches to generate new reagents to study the ESR (Chapter 4). We performed structure-activity relationship studies on sal to improve its activity and characterize its mode of action. We also identified other structurally unrelated compounds that inhibit ER stress-induced apoptosis. Finally, we have undertaken a pilot genetic screen for genes that protect mammalian cells from ER stress. This and future work may contribute significantly to the understanding of ER stress-induced apoptosis and its therapeutic manipulation.