Characterization of mammalian ceramide synthases

描述（由申请人提供）：神经酰胺和相关鞘脂是生物膜和细胞信使的关键结构组分，参与细胞增殖、分化和多种分化细胞功能、衰老和凋亡的调节。神经酰胺通过三种主要途径形成：鞘氨醇酰化为二氢神经酰胺，然后去饱和或羟基化;鞘氨醇酰化;以及更复杂的鞘脂如鞘磷脂的水解。这项资助的重点是负责鞘氨醇碱酰化的酶（称为神经酰胺酶），我们最近发现了三个哺乳动物基因（称为LASS 1，4和5的长寿保证基因同源物），每个编码（二氢）神经酰胺酶，产生特定的分子亚种（二氢）-神经酰胺由于高度选择性的脂肪酰辅酶A共底物。在这项研究中，我们将进一步描述这些以及其他哺乳动物LASS同系物，以确定每个在鞘脂合成中的作用。这些研究将采用多种方法的组合：使用基因克隆来表达，RNAi来抑制感兴趣的基因;神经酰胺合酶活性的生物化学表征;荧光显微镜来探索亚细胞定位;液相色谱、电喷雾串联质谱（MS/MS和MS/MS/MS）用于神经酰胺亚种、复合鞘脂和其他感兴趣的代谢物（例如，鞘氨醇碱1-磷酸盐等）。质谱分析也将用于使用稳定同位素标记的骨架定量特定亚种的生物合成和周转。这些工具还将用于探索神经酰胺生物合成如何在特定LASS家族成员的mRNA表达水平上进行调节，其中鞘脂代谢在遗传上受到干扰和（或）通过添加外源鞘脂和类似物（包括药物safingol和FTY 720）。这些研究将提供关于鞘脂代谢的基本信息，并且所选择的实验系统将提供对神经酰胺脱氢酶在诸如癌症的疾病中的作用的深入了解，其中已知LASS 1表达异常以及神经酰胺合酶参与基于鞘脂的治疗剂的作用。

DESCRIPTION (provided by applicant): Ceramides and related sphingolipids are key structural components of biological membranes and cell messengers involved in the regulation of cell proliferation, differentiation and numerous differentiated cell functions, senescence and apoptosis. Ceramide is formed by three major pathways: acylation of sphinganine to dihydroceramides followed by desaturation or hydroxylation; acylation of sphingosine; and hydrolysis of more complex sphingolipids, such as sphingomyelin. This grant focuses on the enzymes responsible for acylation of sphingoid bases (termed ceramide synthases) for which we have recently identified three mammalian genes (termed LASS1, 4 and 5 for longevity assurance gene homologs) that each code for (dihydro)ceramide synthases that produces specific molecular subspecies of (dihydro)- ceramide due to a high degree of selectivity for the fatty acyl-CoA co-substrate. In this grant we will further characterize these as well as additional mammalian LASS homologs to determine the roles of each in sphingolipid synthesis. The studies will employ a combination of approaches: use of gene cloning to express, and RNAi to suppress, the genes of interest; biochemical characterization of the ceramide synthase activities; fluorescence microscopy to explore subcellular localization; and liquid chromatography, electrospray ionization tandem mass spectrometry (MS/MS and MS/MS/MS) for in-depth, quantitative analyses of ceramide subspecies, complex sphingolipids and other metabolites of interest (e.g., sphingoid base 1-phosphates, etc.). Mass spectrometric analyses will also be used to quantify the biosynthesis and turnover of specific subspecies using stable isotope labeled backbones. These tools will also be used to explore how ceramide biosynthesis is regulated at the levels of expression of the mRNA for specific LASS family members under conditions where sphingolipid metabolism has been perturbed genetically and (or) by addition of exogenous sphingolipids and analogs, which include the drugs safingol and FTY720. These studies will provide fundamental information about sphingolipid metabolism, and the selected experimental systems will provide insight into the roles of ceramide synthases in diseases such as cancer where there are known abnormalities in LASS1 expression as well as an involvement of ceramide synthase(s) in the action of sphingolipid-based therapeutics.