Inositol Lipid Regulation of Membrane Fusion & Fission

 描述(申请人提供)：拟议研究的长期目标是确定磷脂酰肌醇(3，5)-双磷酸(PI3，5P2)水平是如何动态调节的。PI3、5P2是一种丰度很低但必不可少的细胞调节因子，在特定的时间以特定的膜的形式瞬时合成。来自我们和其他人的新研究表明，PI3，5P2，对正常的人类生理至关重要。一种预计会降低PI3，5P2的突变会导致一种严重的先天性疾病，导致婴儿死亡，并对多个组织产生严重的病理影响。微小的突变预测会导致轻微的缺陷 动态调节PI3、5P2水平的能力是一种严重的CMT4J神经病变以及一些肌萎缩侧索硬化症(ALS)的基础。另一种微小的突变与癫痫、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PI3，5P2所需的蛋白质对调节突触活动至关重要。这些发现强调了确定PI3、5P2水平如何动态调节的重要性。我们以前的研究表明，PI3，5P2的动态调节是通过一个大的蛋白质复合体来实现的，其中包括一个在酵母中被称为FAB1的保守的脂蛋白激酶，在哺乳动物中的PIKfyve，以及两个保守的调节蛋白Vac14和Fig4。根据ITS序列和体外研究，推测图4提供了PI3，5P2的主要周转途径。然而，我们的研究表明，Fig4的一个主要功能是激活FAB1/PIKfyve；这种激活的机制尚不清楚。此外，激活PIKfyve/FAB1-Vac14-Fig4复合体的上游信号通路尚不清楚，这在确定PI3、5P2水平如何动态调节方面存在另一个关键缺口。我们将通过以下具体目标解决这些差距：1)。确定图4在FAB1/PIKfyve活性调节中的作用；2)确定FAB1激酶活性是否部分通过分子内抑制域调节；3)。确定FAB1和Vac7的磷酸化是否是Pho85-Pho80调节PI3、5P2水平的主要途径，以及CDK5-p35是否是PIKfyve的上游调节因子。这些研究的结果将为调节PI35P2的机制提供重要的见解，并可能为开发治疗特定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.