Study of caspases in apoptosis, metabolism, ageing and cancer
Caspases are cysteine proteases that act as the initiators and executioners of apoptosis. Having cloned one of the first caspases (caspase-2), our laboratory has an ongoing interest in understanding caspase regulation and function. Caspase-2 has been shown to function both as an initiator and effector caspase and is activated by various apoptotic signals. Our current focus is to delineate the molecular mechanisms of caspase-2 function.
Our recent work has found roles for caspase-2 in both apoptotic and non-apoptotic signalling pathways including tumour suppression and ageing. We have demonstrated that loss of caspase-2 enhances tumour formation in a number of mouse models. Furthermore, we have shown that primary and tumour cells from caspase-2 knockout mice show increased genetic instability and aneuploidy. In addition, we and others have shown that caspase-2 deficiency leads to oxidative stress-induced premature aging in mice, as well as metabolic defects.
We are now using various proteomic and bioinformatics approaches to identify specific substrates of caspase-2. We are also using cell and molecular biology techniques along with mouse models to understand how the loss of caspase-2 contributes to tumorigenesis, altered metabolism and ageing-related traits.
Understanding the mechanisms and regulation of developmentally programmed cell death
In addition to mammalian systems we use Drosophila melanogaster as a model system to examine regulation of cell death during animal development. Many of the cell death pathway components found in mammals are conserved in Drosophila, making it a useful genetic system for biological and disease model studies. During Drosophila development programmed cell death of specific tissue, including larval midgut and salivary glands, is triggered in response to a rise in the steroid hormone ecdysone. We discovered that the caspase-dependent apoptotic pathway, which is essential for most programmed cell death, is largely redundant for cell death in midgut. We have also found that the inhibition of autophagy, delays removal of the midgut indicating a role for autophagy as an alternative cell death mechanism in the midgut. Given that the role of autophagy in cell death is a matter of extensive debate, our discovery that midgut cell death can be delayed by genetically blocking autophagy provides a unique model for delineating this controversy. In further studies examining the transcriptional control of programmed cell death we have found that the histone demethylase dUTX is required for the correct spatial and temporal expression of cell death genes during salivary gland removal.
In current projects we are examining the role of autophagy in midgut cell death and determine how the cell growth and death signals are integrated. We also aim to understand the transcriptional regulation of cell death genes in response to ecdysone. We use a range of cellular, molecular and genetic approaches in these projects. Ultimately our goal is to understand the various ways cells die and how these relates to disease.
Role of the Nedd4 family of ubiquitin ligases in physiology and disease
Aberrations in the ubiquitin system underpin the pathogenesis of many diseases including malignancies, neurodegenerative disorders and channelopathies. Ubiquitin-protein ligases (E3s) determine the substrate specificity of the ubiquitination process. The Nedd4 family of E3s, with Nedd4 as the prototypic member (discovered in our laboratory), is evolutionarily conserved and required for the regulation of a number of key signalling pathways. Members of the Nedd4-family also ubiquitinate and control a range of membrane proteins, resulting in their internalisation and degradation.
We have found that Nedd4 deficiency in mouse leads to a number of different phenotypes, including dramatic inhibition of foetal growth due to impaired IGF1 signaling. A closely related protein Nedd4-2 regulates a number of ion channels, including the epithelial sodium channel (ENaC). ENaC is required for sodium absorption across a range of epithelial tissues such as the lung, colon and kidney and is an important regulator of blood sodium concentration and blood pressure. Nedd4-2 knockout also results in deregulated voltage-gated sodium channel function and other pathologies that are the focus of current studies.
Study of adaptor proteins that facilitate and regulate the functions of Nedd4 family members
We have identified a number of Nedd4-interacting proteins (Ndfips) that provide specificity and regulatory complexity to the ubiquitination system. We have found that the primary non-heme iron transporter DMT1 is down-regulated by Ndfip1. Ndfip1 recruits Nedd4 ubiquitin ligases to enable ubiquitination and subsequent degradation of DMT1 via the proteasome/lysosomal systems. This is significant physiologically as Ndfip1-/- mice show significant misregulation of iron homeostasis, along with severe inflammatory disease. Our current focus is to further characterise Ndfip1 and other potential adaptors of the Nedd4 family to provide better understanding of ubiquitin-dependent mechanisms of regulating iron transport and inflammatory disease.