click to enlarge

Essentially all animal cells have the ability to kill themselves by activating a gene-encoded cell suicide program. The execution of this self-destruction program leads to a morphologically distinct form of cell death termed apoptosis. Apoptosis plays an important role in sculpting the developing organism and eliminating unwanted and potentially dangerous cells throughout life. The decision of whether a particular cell will live or die is tightly regulated by many different signals originating both from within the cell and from its environment, and abnormal regulation of apoptosis is associated with a variety of diseases, including cancer, autoimmune diseases, stroke and neurodegenerative disorders. The overall goal of our research is to elucidate the precise mechanism by which cells undergo apoptosis, and how this process is regulated by diverse signaling pathways.

We are using a multidisciplinary approach that integrates Drosophila genetics and molecular biology with mammalian cell culture experiments and biochemical and structural studies. Most of our work utilizes a highly accessible model organism, the fruit fly Drosophila, which offers unique advantages for the discovery of novel cell death genes using powerful genetic techniques. From surveying a large fraction of the Drosophila genome for genes that are required for programmed cell, three apoptotic activators, termed reaper, head involution defective (hid), and grim were identified. All three genes are necessary and sufficient for the activation of apoptosis in Drosophila. Significantly, reaper, hid and grim are all transcriptionally regulated by a variety of death-inducing stimuli, including steroid hormones, segmentation and patterning genes, and DNA damaging agents. Therefore, it appears that these genes act as integrators for relaying different apoptotic signals to the core death program .

Core Death Program, click to enlarge

In order to gain further insight into the mechanism by which reaper hid and grim induce apoptosis, we have designed very sensitive and powerful genetic screens to identify additional cell death genes in Drosophila. These studies have revealed that the cell-killing activity of the HID protein is inactivated upon phosphorylation by MAPK, and this provides an explanation for how survival signals acting through the Ras/MAPK pathway can suppress the induction of apoptosis. We have also isolated and characterized mutations in the Drosophila inhibitor of apoptosis protein-1(diap1) gene, and this work demonstrated that Reaper, Hid and Grim kill by inhibiting the anti-apoptotic activity of Diap1. In both insects and mammals, Inhibitor of Apoptosis Proteins (IAPs) can block cell death by inhibiting caspases, a family of proteases that are key executioners of apoptosis. The active forms of Reaper, Hid and Grim can bind to IAPs and prevent them from caspase inhibition. Many IAPs, including Diap1, have a ubiquitin ligase motif which can be used to target proteins for degradation via the proteosome pathway. We recently found that Reaper, but not Hid, promotes auto-ubiquitination and self-destruction of Diap1. These results suggest a novel strategy for the selective elimination of tumor cells that express elevated IAP protein levels.

We have also characterized a Drosophila homolog of ced-4/Apaf-1, termed hac-1 (for homolog of Apaf-1 and ced-4). Hac-1 is structurally and functionally very similar to Apaf-1 and is required for normal cell death during embryonic development in Drosophila. The expression of hac-1 is regulated both in development and is also induced by ionizing radiation. Our results demonstrate that death-inducing stimuli can simultaneously activate two distinct regulatory pathways controlling caspase activation in Drosophila. We believe that such a dual regulatory input on caspase activation operates also during the control of apoptosis in mammalian cells.

Certain cells, including terminally differentiated neurons in Drosophila, are highly resistant towards cell death. By using Drosophila photoreceptor development as a model system, we hope to gain insights into the survival mechanisms employed for resistance to apoptosis (see more detailed research projects). Since the mechanism of apoptosis has been conserved in evolution from worms to insects to man, knowledge gained from studying cell death in Drosophila is likely to apply to mammalian systems as well. In order to test this directly, we are studying the activity of Drosophila cell death genes and their mouse or human homologues in cultured mammalian cells. We are also interested in biochemical and structural studies of selected cell death proteins in order to determine their precise mechanism of action. We expect that knowledge gained from this work can ultimately be exploited to manipulate apoptosis for therapeutic benefits.