Inositol Lipid Regulation of Membrane Fusion & Fission

 描述（由申请方提供）：拟定研究的长期目标是确定磷脂酰肌醇（3，5）-二磷酸（PI 3，5 P2）水平如何动态调节。PI 3，5 P2是一种丰度非常低但必需的细胞调节剂，在特定时间瞬时合成为特定膜。我们和其他人的新兴研究表明，PI 3，5 P2对正常的人体生理至关重要。预测降低PI 3，5 P2的突变导致严重的先天性疾病，导致婴儿死亡，并对多种组织具有严重的病理影响。预测微小突变会导致 动态调节PI 3，5 P2水平能力是严重形式的CMT 4J（一种神经病）以及某些肌萎缩侧索硬化（ALS）病例的基础。另一个微小的突变与癫痫、大脑发育异常和严重的精神问题有关。值得注意的是，我们发现调节PI 3、5 P2所需的蛋白质对于调节突触活动至关重要。这些发现强调了确定PI 3，5 P2水平如何动态调节的重要性。我们以前的研究表明，PI 3，5 P2的动态调节是通过一个大的蛋白质复合物发生的，该蛋白质复合物包括酵母中称为Fab 1的保守脂质激酶和哺乳动物中的PIKfyve，以及两个保守的调节蛋白Vac 14和Fig 4。基于其序列和体外研究，假设图4提供了PI 3，5 P2转换的主要途径。然而，我们的研究表明图4的主要功能是激活Fab 1/PIKfyve;这种激活的机制尚不清楚。此外，激活PIKfyve/Fab 1-Vac 14-Fig 4复合物的上游信号传导途径是未知的，这在确定PI 3，5 P2水平如何动态调节方面存在另一个关键差距。我们将通过以下具体目标来解决这些差距：1）。确定图4在调节Fab 1/PIKfyve活性中的作用; 2）确定Fab 1激酶活性是否部分通过分子内抑制结构域调节; 3）.确定Fab 1和Vac 7的磷酸化是否是Pho 85-Pho 80调节PI 3，5 P2水平的主要途径，以及Cdk 5-p35是否是PIKfyve的上游调节因子。这些研究的结果将为调节PI 35 P2的机制提供重要的见解，并可能为开发治疗选定的神经和精神疾病的治疗策略提供新的途径。

 DESCRIPTION (provided by applicant): The long range goal of the proposed studies is to determine how phosphatidylinositol (3, 5)-bis phosphate (PI3, 5P2) levels are dynamically regulated. PI3, 5P2, a very low abundance, yet essential cellular regulator, is transiently synthesized as specific membranes at specific times. Emerging studies from us and others indicate that PI3, 5P2, is critical for normal human physiology. A mutation predicted to lower PI3, 5P2 causes a severe congenital disorder that results in infant mortality and has severe pathological effects on multiple tissues. Minor mutations predicted to result in a modest defect in the ability to dynamically regulate PI3, 5P2 levels underlie a severe form of CMT4J, a neuropathy, as well as some cases of amyotrophic lateral sclerosis (ALS). Another minor mutation has been linked to epilepsy, abnormalities in brain development and severe psychiatric problems. Notably, we discovered that proteins required for the regulation of PI3, 5P2 are critical for modulation of synaptic activity. These findings underscore the importance of determining how PI3, 5P2 levels are dynamically regulated. Our previous studies revealed that the dynamic regulation of PI3, 5P2 occurs through a large protein complex which includes a conserved lipid kinase called Fab1 in yeast, and PIKfyve in mammals, as well as two conserved regulatory proteins, Vac14 and Fig4. Based on its sequence, and in vitro studies, it was assumed that Fig4 provides the major route for turnover of PI3, 5P2. However, our studies suggest that a major function of Fig4 is in the activation of Fab1/PIKfyve; the mechanism of this activation is unknown. In addition the upstream signaling pathways that activate the PIKfyve/Fab1-Vac14-Fig4 complex are unknown, and this presents another critical gap in determining how PI3, 5P2 levels are dynamically regulated. We will address these gaps with the following specific aims: 1). Determine roles of Fig4 in the regulation of Fab1/PIKfyve activity; 2) Determine whether Fab1 kinase activity is regulated in part via an intramolecular inhibitory domain; 3). Determine whether phosphorylation of Fab1 and Vac7 is the major pathway for Pho85- Pho80 regulation of PI3, 5P2 levels, and whether Cdk5-p35 is an upstream regulator of PIKfyve. Results of these studies will provide significant insights into the mechanisms that regulate PI35P2, and may provide new avenues to develop therapeutic strategies to treat selected neurological and psychiatric disorders.