Cell death is widespread during the development of the nervous system, where it helps to ensure that the proper number and types of connections are formed between neurons and their targets. It is often initiated when neurons fail to receive adequate survival promoting signals from trophic factors such as nerve growth factor (NGF). Recent evidence suggests that a similar type of death may occur in human neuronal disorders and degenerative diseases, when neurons gain insufficient access to trophic support. Trophic factor deprivation-induced death requires RNA and protein synthesis and involves complex, incompletely understood mechanisms leading to activation of caspases that dismantle the cell. We and others have hypothesized that genes upregulated after trophic factor withdrawal function in critical cell death pathways. We previously identified SM-20 as one of a select group of genes whose expression increases in NGF-deprived neurons and that promote caspase-dependent death when overexpressed. Very recently, SM-20 and two closely related proteins (collectively called EGLN proteins) were shown to comprise a new family of prolyl hydroxylases involved in regulating the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). EGLN-catalyzed proline hydroxylation destabilizes HIF-1alpha by increasing its affinity for a ubiquitin ligase. Based on these and other findings, we hypothesize that SM-20/EGLN3-mediated proline hydroxylation of critical target proteins (including but not limited to HIF-1alpha) plays an important role in regulating cell death initiated by trophic factor withdrawal. Here we propose experiments to (1) define the importance of SM-20/EGLN3 for trophic factor deprivation-induced death, and (2) to characterize its mechanism of action in NGF-deprived neurons.
In aim 1, we shall assess the importance of SM-20/EGLN3 expression and activity for death caused by NGF withdrawal.
For aim 2, we shall use complementary approaches to determine if HIF-1alpha transcription factor activity is regulated by the presence or absence of NGF and if stabilized, SM-20/EGLN3-resistant forms of HIF-1alpha are neuroprotective. Additional experiments will determine the effects of disrupting HIF-1alpha expression on trophic factor deprivation-induced death and NGF-dependent survival. In the third aim, we will examine the functional significance of a recently identified interaction between SM-20/EGLN3 and NRAGE, a protein previously shown to bind to the NGF receptor p75 NTR. Lastly, in aim 4 we propose new approaches for identifying novel substrates for this increasingly important family of enzymes. These studies should help further our understanding of the mechanisms that lead to trophic factor deprivation-induced death. They will also provide new information concerning the function of EGLN-catalyzed proline hydroxylation as a novel mechanism for altering protein function in neurons.
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