Abstract 15606: Genome-wide Loss-of-Function Screen Identifies SNARE Proteins as a Central Component of Programmed Necrosis
Background: It has become apparent that while apoptosis occurs in the heart after myocardial infarction, more cells die by necrosis, which we and others have recently shown can be a regulated process. Defining the molecular mechanisms underlying programmed cellular necrosis may thus offer novel treatment strategies for cardiovascular disease and other cell death-driven disease processes.
Methods: To identify novel molecular effectors of programmed necrotic cell death we developed a functional cell-based assay to quantitate plasma membrane rupture (a hallmark of necrosis) in conjunction with a high-throughput, robotic-based screen using an shRNA total genome library in lentivirus. This library consists of over 77,500 clones targeting more than 16,000 genes in the mouse genome (~5 clones/gene target) to enable loss-of-function screening in a 96-well format.
Results: We identified hundreds of shRNA clones that enhanced membrane rupture (protective gene targets), as well as hundreds of shRNA clones that inhibited membrane rupture (pro-necrosis gene targets). Using bioinformatic enrichment analyses and gene ontology grouping, a novel and interesting pathway emerged that was associated with the SNARE-complex and genes affecting membrane fusion events. Secondary screening with a custom SNARE pathway library of 350 lenti-shRNAs targeted against 66 genes confirmed a prominent role in programmed necrosis initiated by a variety of death-inducing stimuli. Indeed, knockdown of select genes in this membrane fusion/SNARE pathway ameliorated programmed cellular necrosis in vitro.
Summary: These findings suggest membrane fusion/SNARE complex pathways as a novel biological component of programmed necrosis and provide an entirely new therapeutic strategy for reducing cell death in the heart following myocardial infarction injury.
- © 2013 by American Heart Association, Inc.