Intracellular Target of Sphingosine-1-Phosphate

描述(由申请人提供)： 鞘氨醇-1-磷酸(S1P)是一种有效的生物活性脂质，在脑发育、神经元存活和神经系统重建中发挥关键作用。神经鞘氨醇激酶(SphKs)在神经元中形成SIP受多种激动剂的刺激，包括神经生长因子(NGF)和神经营养素-3(NT-3)，其许多功能是由其细胞表面受体(S1P1-5)介导的。然而，越来越多的证据表明，S1P也具有受体非依赖性的细胞内效应，特别是那些与神经元存活相关的效应。阻碍了解S1P在细胞内的作用的一个关键因素是缺乏真正的细胞内靶点的识别。产生S1P的两种SphK同工酶SphK1和SphK2的细胞定位已经开始提供一些线索。SphK1主要是胞浆的，在多种刺激下被激活并转移到质膜上，并转移到其受体附近。相反，在许多类型的细胞中，SphK2主要存在于其功能未知的细胞核中。我的初步数据表明，S1P确实与至少一种存在于细胞核中的蛋白质结合，而且它还可以改变几种转录因子的DMA结合活性。这一提议的主要假设是S1P与介导其细胞内和/或核内活动的特定细胞内靶点相互作用。在细胞核中产生的S1P可能通过与转录因子或其他一些调节因子结合来调节基因的表达，本建议的目的是阐明S1P的直接细胞内靶标。为此，我将鉴定与S1P结合的细胞内蛋白，并使用允许多个转录因子同时活性分析的转录因子活性膜来研究S1P对转录因子DNA结合活性的影响。此外，我将确定SphK1或SphK2在细胞内产生的S1P是否调节不同的转录因子活性。最后，S1P的特定细胞位置的意义将通过改变定位于不同亚细胞中的两种鞘氨醇激酶同工酶的表达来确定。这将通过用小干扰RNA下调每一种激酶的表达，并观察其对转录因子活性和基因表达的影响来实现。细胞内S1P增强神经元存活并抑制细胞死亡，这是中风、阿尔茨海默氏症和帕金森氏症等神经退行性疾病的关键过程。破译这种鞘糖脂代谢产物的作用机制，可以为防治人类毁灭性的神经退行性疾病提供线索。

DESCRIPTION (provided by applicant): Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that plays key roles in brain development, neuronal survival, and nervous system remodeling. Formation of SIP in neurons by sphingosine kinases (SphKs) is stimulated by a variety of agonists, including nerve growth factor (NGF) and neurotrophin-3 (NT- 3), and many of its functions are mediated by its cell surface receptors (S1P1-5). However, there is accumulating evidence suggesting that S1P also has receptor-independent intracellular effects, particularly those related to neuronal survival. A key factor hindering understanding of the intracellular roles of S1P is the lack of identification of bona fide intracellular targets. Cellular localizations of the two SphK isoenzymes that produce S1P, SphK1 and SphK2, have begun to provide some clues. SphK1 is mainly cytosolic and is activated and translocated to the plasma membrane to the vicinity of its receptors by many stimuli. In contrast, in many cell types, SphK2 is mainly found in the nucleus where its function is not known. My preliminary data indicate that S1P does bind to at least one protein that is present in the nucleus, and that it can also alter the DMA binding activity of several transcription factors. The main hypothesis of this proposal is that S1P interacts with specific intracellular targets that mediate its intracellular and/or intranuclear actions. S1P produced in the nucleus may modulate gene expression by binding to a transcription factor or to some other regulator, and the objective of this proposal is the elucidation of the direct intracellular targets of S1 P. To this end, I will identify intracellular proteins that bind S1P and investigate the effect of S1P on transcription factor DNA binding activity using a transcription factor activity membrane that allows simultaneous activity profiling of multiple transcription factors. In addition, I will determine whether intracellularly generated S1P by SphK1 or SphK2 modulates distinct transcription factor activities. Finally, the significance of the specific cellular location of S1P will be determined by altering the expression of the two sphingosine kinase isoenzymes, which are localized in different subcellular compartments. This will be accomplished by downregulation of expression of each of the kinases with small interfering RNA, and observing the effects on transcription factor activity and gene expression. Intracellular S1P enhances neuronal survival and suppresses cell death, key processes involved in neurodegenerative disorders such as stroke and Alzheimer's and Parkinson's diseases. Deciphering the mechanism of action of this sphingolipid metabolite could provide clues for the prevention and treatment of devastating human neurodegenerative diseases.