Final Report Summary - DEATHSWITCHING (Identifying genes and pathways that drive molecular switches and back-up mechanisms between apoptosis and autophagy)
The first major goal of the project was to study how the cell’s built-in survival and programmed cell death pathways are interconnected. Specifically, we wished to understand how the cell switches between autophagy, mainly a survival pathway, and cell death by apoptosis. We successfully developed and used a protein-protein interaction (PPI) platform to identify in a global manner novel interactions within and between the apoptosis and autophagy pathways. The platform comprised 63 apoptotic and autophagic proteins and led to the identification of 46 novel PPIs, including 9 direct interactions between autophagic and apoptotic proteins. This platform also enabled the monitoring of simultaneous changes in PPIs in real time, in cells. Among the newly identified interactions, we studied in-depth candidate proteins interacting with DAPK2, a Ser/Thr kinase that can be directed to apoptosis or autophagy depending on its interacting proteins. The study of the DAPK2 interaction with14-3-3 revealed a new mechanism that blocks the cellular activity of this critical kinase. Study of another identified interacting protein, Atg14, led to the elucidation of the mode of action of DAPK2 in autophagy, mapping it within the Atg14/Vps34/Beclin-1 complex. A long-term research project that developed from this line of investigation showed that DAPK2 is activated by AMPK during metabolic stress, thereby triggering the initial stages of autophagy through phosphorylation of Beclin-1. We also applied ‘educated guess’ approaches in parallel to the global PPI screens to characterize the molecular signature of the ‘deathswitching’ process. To this end, we identified changes in the expression of key apoptosis proteins that contribute to the switch between survival autophagy and apoptosis. Another direction included the study of how phosphorylation of the autophagic protein Atg12, previously shown to exert a dual function in autophagy and apoptosis, affects either of its functions.
A second major goal of the project was to understand how, under specific circumstances, autophagy, usually a survival pathway, switches to a lethal mechanism for elimination of the cell by over self-consumption. To this end, we conducted a signalome-wide shRNA screen to identify molecular regulators of resveratrol-induced autophagic cell death. We established the criteria of autophagic cell death, comprising enhanced autophagy flux in the absence of alternative death pathways, such as apoptosis and necrosis, and accumulation/activation of autolysosomes consuming the cytosol, internal membranes and organelles. Among the candidate genes emerging from the screen was GBA1, which encodes a lysosomal enzyme responsible for generation of ceramide. Our data support the hypothesis that changes in ceramide metabolism sustain a high autophagy flux that hyper-consumes cellular content, rendering it lethal. Moreover, the Drosophila orthologue, Gba1a, is necessary for developmental autophagic cell death in the larval midgut. Thus we have made significant progress in understanding how autophagy interconnects with cell death in different scenarios. Finally, we have studied the contribution of cell death to proamniotic cavity formation in the early mouse embryo, representing the first wave of programmed cell death during development. We identified the contribution of canonical intrinsic apoptotic pathways, and in parallel, alternative caspase-independent death programs, to the process. Thus, the concept of cross-interactions and molecular switches between different cell modalities, initially studied in cell lines as a convenient model, is relevant to developmental programs.
A second major goal of the project was to understand how, under specific circumstances, autophagy, usually a survival pathway, switches to a lethal mechanism for elimination of the cell by over self-consumption. To this end, we conducted a signalome-wide shRNA screen to identify molecular regulators of resveratrol-induced autophagic cell death. We established the criteria of autophagic cell death, comprising enhanced autophagy flux in the absence of alternative death pathways, such as apoptosis and necrosis, and accumulation/activation of autolysosomes consuming the cytosol, internal membranes and organelles. Among the candidate genes emerging from the screen was GBA1, which encodes a lysosomal enzyme responsible for generation of ceramide. Our data support the hypothesis that changes in ceramide metabolism sustain a high autophagy flux that hyper-consumes cellular content, rendering it lethal. Moreover, the Drosophila orthologue, Gba1a, is necessary for developmental autophagic cell death in the larval midgut. Thus we have made significant progress in understanding how autophagy interconnects with cell death in different scenarios. Finally, we have studied the contribution of cell death to proamniotic cavity formation in the early mouse embryo, representing the first wave of programmed cell death during development. We identified the contribution of canonical intrinsic apoptotic pathways, and in parallel, alternative caspase-independent death programs, to the process. Thus, the concept of cross-interactions and molecular switches between different cell modalities, initially studied in cell lines as a convenient model, is relevant to developmental programs.