Characterization of mammalian ceramide synthases

描述(申请人提供)：神经酰胺和相关的鞘磷脂是生物膜和细胞信使的关键结构成分，参与调节细胞的增殖、分化和许多分化的细胞功能、衰老和凋亡。神经酰胺的形成有三条主要途径：神经鞘氨酸酰化为二氢神经酰胺，然后脱饱和或羟化；神经鞘氨醇的酰化；以及更复杂的鞘磷脂的水解性，如鞘磷脂。这笔赠款主要用于负责狮身人面像碱基的酰化反应的酶(称为神经酰胺合成酶)，我们最近为这些酶确定了三个哺乳动物基因(称为LASS1、4和5，用于长寿保证基因同源物)，每个基因都编码(二氢)神经酰胺合成酶，由于对脂肪酰基-辅酶A共底物具有高度的选择性，(二氢)神经酰胺合成酶产生特定的分子亚种(二氢)-神经酰胺。在这项资助中，我们将进一步表征这些同系物以及其他哺乳动物LASS同源物，以确定每种同源物在神经鞘脂脂合成中的作用。这些研究将采用多种方法相结合的方法：使用基因克隆来表达感兴趣的基因，并使用RNAi来抑制感兴趣的基因；神经酰胺合成酶活性的生化特征；荧光显微镜来探索亚细胞定位；以及液相色谱、电喷雾电离串联质谱仪(MS/MS和MS/MS/MS)来深入、定量地分析神经酰胺亚种、复杂的鞘磷脂和其他感兴趣的代谢物(如狮身人面像碱基1-磷酸盐等)。质谱分析也将被用来使用稳定同位素标记的骨架来量化特定亚种的生物合成和周转。这些工具还将用于探索在鞘脂代谢受到遗传干扰和(或)添加外源性鞘脂及其类似物(包括药物safingol和FTY720)的情况下，神经酰胺生物合成是如何在特定LASs家族成员的mRNA表达水平上进行调节的。这些研究将提供有关鞘磷脂代谢的基本信息，所选的实验系统将提供神经酰胺合成酶在已知LASS1表达异常的疾病中的作用，以及神经酰胺合成酶(S)在基于鞘磷脂的疗法中的作用。

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.