Genes That Regulate Neuronal Cell Death

描述(申请人提供)：细胞死亡在神经系统发育过程中广泛存在，它有助于确保神经元和它们的目标之间形成适当数量和类型的连接。它通常是在神经元未能从神经生长因子(NGF)等营养因子那里获得足够的促进生存信号时启动的。最近的证据表明，当神经元获得的营养支持不足时，人类神经元紊乱和退行性疾病可能会发生类似类型的死亡。营养因子剥夺诱导的死亡需要RNA和蛋白质的合成，并涉及复杂的、尚未完全了解的导致caspase激活的机制，从而分解细胞。我们和其他人假设，在营养因子停用后，基因上调在关键细胞死亡途径中发挥作用。我们之前发现SM-20是一组精选的基因之一，其在NGF剥夺的神经元中表达增加，并在过度表达时促进caspase依赖的死亡。最近，SM-20和两个密切相关的蛋白(统称为EGLN蛋白)被发现组成了一个新的Pro羟基酶家族，参与调控转录因子缺氧诱导因子1α(HIF-1α)。EGLN催化的脯氨酸羟化通过增加其与泛素连接酶的亲和力来破坏HIF-1α的稳定。基于这些和其他发现，我们推测SM-20/EGLN3介导的关键靶蛋白(包括但不限于HIF-1α)的脯氨酸羟化在调节营养因子停用引发的细胞死亡中发挥重要作用。在这里，我们建议进行实验，以(1)确定SM-20/EGLN3在营养因子剥夺诱导的死亡中的重要性，以及(2)表征其在NGF剥夺神经元中的作用机制。在目标1中，我们将评估SM-20/EGLN3的表达和活性在NGF戒断引起的死亡中的重要性。对于目标2，我们将使用互补的方法来确定HIF-1α转录因子的活性是否受NGF的存在或不存在的调节，如果稳定，SM-20/EGLN3抗性形式的HIF-1α具有神经保护作用。其他实验将确定干扰HIF-1α表达对营养因子剥夺诱导的死亡和NGF依赖生存的影响。在第三个目标中，我们将研究最近发现的SM-20/EGLN3与NRAGE之间的相互作用的功能意义。NRAGE是一种先前被证明与NGF受体p75 NTR结合的蛋白质。最后，在目标4中，我们提出了为这一日益重要的酶家族确定新底物的新方法。这些研究将有助于我们进一步了解营养因子剥夺导致死亡的机制。他们还将提供关于EGLN催化的脯氨酸羟化作为改变神经元蛋白质功能的新机制的新信息。

DESCRIPTION (provided by applicant): 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.