Intracellular Target of Sphingosine-1-Phosphate

1-磷酸鞘氨醇的细胞内靶标

• 批准号: 7624345
• 负责人: GRAHAM M STRUB
• 金额: $ 3.44万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-18 至 2011-06-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7624345
• 关键词: Affect; Agonist; Alzheimer' s Disease; Anabolism; Apoptosis; Apoptotic; Binding; Blood Vessels; Brain; Brain region; Breast Cancer Cell; Caspase; Cell Cycle Progression; Cell Death; Cell Nucleus; Cell Surface Receptors; Cell division; Cell membrane; Cell physiology; Cells; Ceramides; Complement; Consensus Sequence; DNA; DNA Binding; Data; Development; Down-Regulation; EGF gene; Electrophoretic Mobility Shift Assay; Elements; Embryo; Engineering; Equilibrium; Exhibits; Family; Fibroblasts; G-Protein-Coupled Receptors; Gene Expression; Genes; Genetic Transcription; Growth; Growth Factor; Histone Acetylation; Histone Deacetylase; Human; Isoenzymes; Knock-out; Knockout Mice; Label; Lentivirus Vector; Lipids; Location; Lymphocyte; MCF7 cell; Mammalian Cell; Mass Spectrum Analysis; Mediating; Membrane Proteins; Mitosis; Modeling; Multiple Sclerosis; Mus; NTF3 gene; Nerve Growth Factors; Nervous system structure; Neural Tube Closure; Neurodegenerative Disorders; Neuroepithelial; Neuroglia; Neurons; Neurotrophin 3; Nuclear Extract; Nuclear Protein; Nuclear Proteins; PC12 Cells; Parkinson Disease; Pheochromocytoma; Phosphate-Binding Proteins; Phosphotransferases; Plants; Platelet-Derived Growth Factor; Play; Polyacrylamide Gel Electrophoresis; Prevention; Process; Production; Protein Binding; Proteins; Rattus; Regulation; Role; SPHK1 enzyme; Sepharose; Small Interfering RNA; Sphingolipids; Sphingosine; Sphingosine-1-Phosphate Receptor; Spinal Ganglia; Stimulus; Stroke; Telencephalon; Vascular Endothelial Growth Factors; Yeasts; angiogenesis; biological adaptation to stress; cell growth; cell type; gene repression; interest; membrane activity; migration; nervous system disorder; neuroepithelium; neurogenesis; neuron loss; neuronal survival; novel; overexpression; pi bond; polyacrylamide gels; prohibitin; receptor; relating to nervous system; response; small hairpin RNA; sphingosine 1-phosphate; sphingosine kinase; sphingosine kinase type 2; sphingosine-1-phosphate lyase; sphingosine-1-phosphate phosphatase; stress activated protein kinase; trafficking; transcription factor

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.

描述（由申请人提供）： 1-磷酸鞘氨醇 (S1P) 是一种有效的生物活性脂质，在大脑发育、神经元存活和神经系统重塑中发挥着关键作用。神经元中鞘氨醇激酶 (SphK) 形成 SIP 受到多种激动剂的刺激，包括神经生长因子 (NGF) 和神经营养蛋白-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 还具有受体独立的细胞内效应，特别是与神经元存活相关的效应。阻碍了解 S1P 细胞内作用的一个关键因素是缺乏真正的细胞内靶标的识别。产生 S1P 的两种 SphK 同工酶（SphK1 和 SphK2）的细胞定位已开始提供一些线索。 SphK1 主要存在于细胞质中，在许多刺激下被激活并转移至质膜至其受体附近。相比之下，在许多细胞类型中，SphK2 主要存在于细胞核中，但其功能尚不清楚。我的初步数据表明，S1P 确实与细胞核中存在的至少一种蛋白质结合，并且它还可以改变几种转录因子的 DMA 结合活性。该提议的主要假设是 S1P 与介导其细胞内和/或核内作用的特定细胞内靶标相互作用。细胞核中产生的 S1P 可能通过与转录因子或其他调节因子结合来调节基因表达，本提案的目的是阐明 S1P 的直接细胞内靶标。为此，我将鉴定结合 S1P 的细胞内蛋白，并使用转录因子活性膜研究 S1P 对转录因子 DNA 结合活性的影响，该转录因子活性膜允许同时分析多个转录因子的活性。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 特定细胞位置的重要性将通过改变位于不同亚细胞区室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下调每种激酶的表达并观察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、阿尔茨海默病和帕金森病等神经退行性疾病中涉及的关键过程。破译这种鞘脂代谢物的作用机制可以为预防和治疗毁灭性的人类神经退行性疾病提供线索。