A systems level strategy for analyzing the cell death network: implication in exploring the apoptosis/autophagy connection

Abstract

The mammalian cell death network comprises three distinct functional modules: apoptosis, autophagy and programmed necrosis. Currently, the field lacks systems level approaches to assess the extent to which the intermodular connectivity affects cell death performance. Here, we developed a platform that is based on single and double sets of RNAi-mediated perturbations targeting combinations of apoptotic and autophagic genes. The outcome of perturbations is measured both at the level of the overall cell death responses, using an unbiased quantitative reporter, and by assessing the molecular responses within the different functional modules. Epistatic analyses determine whether seemingly unrelated pairs of proteins are genetically linked. The initial running of this platform in etoposide-treated cells, using a few single and double perturbations, identified several levels of connectivity between apoptosis and autophagy. The knock down of caspase3 turned on a switch toward autophagic cell death, which requires Atg5 or Beclin-1. In addition, a reciprocal connection between these two autophagic genes and apoptosis was identified. By applying computational tools that are based on mining the protein-protein interaction database, a novel biochemical pathway connecting between Atg5 and caspase3 is suggested. Scaling up this platform into hundreds of perturbations potentially has a wide, general scope of applicability, and will provide the basis for future modeling of the cell death network. The process of programmed cell death (PCD) is driven by a network of proteins connected to each other in an intricate manner. Over the past two decades, many of the network's proteins (nodes) and the interactions among them (edges; mostly post-translation modifications) have been identified. The PCD network is turned on by well-defined input signals, such as activation of death receptors or exposure to DNA damaging agents. The efficiency of its performance determines the individual cell's probability to die, which, when assessed over a large population of cells, can be translated into the percent of cell death. Dying cells can display several distinct cell death phenotypes, each driven by a different subset of proteins and molecular pathways. Examples are the caspase-dependent apoptotic cell death, autophagic cell death and programmed necrosis. 1 Cells exposed to the same input signal can switch from one cell death modality to another in response to specific perturbations, 2-4 and in some cases, a mixed type of cell death can also be observed. [5][6][7] This led us to propose here a working model, according to which the proteins that mediate the three different cell death phenotypes should be integrated within a common network, and the corresponding subsets of proteins should be considered as functional modules within this global network. To study the network as a whole, new strategies capable of analyzing the connectivity within and between the functional modules are required.Here, we developed a platform for dissecting the network's a...

Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak

“…1B) (5,15,16). Because there are conflicting reports claiming autophagy can promote both cell survival and death, we measured the ability of the BH3 mimetic compound ABT-737 to affect cell death either positively or negatively when autophagy was induced by etoposide (5,9,17). After 96 h, etoposide induced moderate cell death in Bax…”

Human ALKBH7 is required for alkylation and oxidation-induced programmed necrosis

“…We treated control and ALKBH7-depleted cell lines with DNA-damaging agents known to induce apoptosis in 293T cells; namely, UV and etoposide (Mourtada-Maarabouni et al 2009;Zalckvar et al 2010). In addition, we tested staurosporine (STS), a well-characterized inducer of apoptosis.…”

ANAT: A Tool for Constructing and Analyzing Functional Protein Networks

“…The resulting anchored network suggests testable hypotheses for the wiring of the previously observed crosstalk between these two cellular processes (48)(49)(50). To illustrate the effectiveness of ANAT, we compared models generated with ANAT to those from six published software tools (11,16,17,19,20,25) 2.…”

“…We previously described how to compute suboptimal solutions (up to x%) for the shortest-path problem (50). In ANAT, this procedure was extended to facilitate margin computation in the general and anchored network inference problems by using suboptimal shortest paths and by modifying the respective inference algorithms.…”

“…Previously, we used a shortest-path computation to infer a single crosstalk pathway connecting caspase-3, a central effector caspase in the apoptotic cascade that is not connected by any known PPI to the autophagic network, and Atg5, an autophagic protein (50). We used the anchored network algorithm of ANAT to extend this work and explore a larger portion of the pathways that connect autophagy with apoptosis in humans.…”