INVESTIGATION OF 1-DEOXYDIHYDROCERAMIDE BIOSYNTHESIS BY MAMMALIAN CELLS

哺乳动物细胞 1-脱氧二氢神经酰胺生物合成的研究

• 批准号: 8365570
• 负责人: ALFRED Harrison MERRILL
• 金额: $ 1.54万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365570
• 关键词: Alanine; Amino Acids; Anabolism; Animals; Biology; Categories; Cell Line; Cells; Ceramides; Coupled; Electrospray Ionization; Funding; Grant; High Pressure Liquid Chromatography; Incubated; Investigation; Mammalian Cell; Mass Spectrum Analysis; Medicine; Methods; National Center for Research Resources; Palmitoyl Coenzyme A; Physical condensation; Play; Principal Investigator; Pulsar; Regulation; Research; Research Infrastructure; Resolution; Resources; Role; Sampling; Serine; Source; Speed; Sphingolipids; Stable Isotope Labeling; Structure; Supplementation; Time; United States National Institutes of Health; base; clinically relevant; cost; interest; mass spectrometer; novel; overexpression; serine palmitoyltransferase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A novel sphingoid base, 1-deoxysphinganine, and its N-acyl-metabolites were recently discovered in studies of mammalian cells in culture and animals (Zitomer et al., J. Biol. Chem. 284:4786, 2009) and represent a novel new category of sphingolipid. 1-Deoxysphinganine was shown to arise from condensation of L-alanine with palmitoyl-CoA by cells in culture, and most is metabolized to 1-deoxydihydroceramides, rather than accumulating as the free sphingoid base. In anticipation of interest in these compounds, such as their possible elevation in clinically relevant samples, we have now developed a high-throughput and structure-specific method for analysis of 1-deoxysphingolipids by high performance liquid chromatography coupled to an electrospray ionization (ESI) QSTAR Pulsar i time-of-flight (TOF) mass spectrometer which combines, speed, sensitivity, high mass accuracy and high resolution. 1-Deoxydihydroceramides were extracted from a Hek293 cell line stably overexpressing serine palmitoyltransferase (SPT1/2 cells) which was incubated with different stable isotope labeled amino acids, such as [3-13C]-L-Serine, [2,3-13C2]-L-Alanine, and [3-D3]-L-Alanine, and examined by LC-ESI MS/MS. These amino acid supplementations show that the novel 1-deoxysphingolipid arises from condensation of alanine with palmitoyl-CoA as evidenced by incorporation of [2,3-13C2]-L-alanine and [3-D3]-L-Alanine into 13C2-1-deoxydihydroceramides and D3-1-deoxydihydroceramides, respectively, by cells in culture. The results show that these underappreciated categories of bioactive sphingoid bases and "ceramides" that are likely to play important roles in cell regulation can be separated and detected by developed LC MS/MS methods.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 最近在培养物和动物的哺乳动物细胞研究中发现了一种新型的鞘类碱，1-脱氧肾上腺素及其N-酰基 - 金代谢物（Zitomer等，J。Biol。Chem。Chem。284：4786，2009），并代表了一种新颖鞘脂的新类别。 1-脱氧肾上腺素显示出是由培养细胞中L-丙氨酸与棕榈酰-COA的凝结产生的，并且大多数被代谢为1-脱氧二氢氧化氧化物酰胺，而不是积聚为游离的鞘氨液碱。为了期待这些化合物的兴趣，例如它们在临床相关样品中的可能升高，我们现在开发了一种高通量和结构特异性方法，用于通过高性能液相色谱与电喷雾电离（ESI）（ESI）耦合的高性能液相色谱法分析1-脱氧脂质脂质。 QSTAR PULSAR I飞行时间（TOF）质谱仪，结合了速度，灵敏度，高质量精度和高分辨率。从HEK293细胞系中提取1-脱氧二氢可酰胺，稳定过表达的丝氨酸棕榈酰转移酶（SPT1/2细胞）与不同稳定的同位素标记的氨基酸（例如[3-13C] -L- l- serine，[2,3-13C2] [2,3-13c] [2,3-13c]）孵育。 -l-丙氨酸和[3-D3] -L-丙氨酸，并通过LC-ESI MS/MS检查。这些补充这些氨基酸表明，新型的1-脱氧胆脂是由丙氨酸与棕榈酰-COA的凝结产生的，这可以通过将[2,3-13c2] -l-丙氨酸和[3-D-D3] -L-丙氨酸掺入13c2-1中证明。 - 脱氧二氢可酰胺和D3-1-脱氧二氢可酰胺分别通过培养细胞中的细胞。结果表明，这些可能在细胞调节中起重要作用的生物活性鞘脂碱和“神经酰胺”的类别可以通过开发的LC MS/MS方法分离和检测。