Characterization of mammalian ceramide synthases

描述（申请人提供）：神经酰胺和相关鞘脂是生物膜的关键结构成分和细胞信使，参与调节细胞增殖、分化和许多分化细胞功能、衰老和凋亡。神经酰胺的形成有三个主要途径：鞘氨酰化为二氢神经酰胺，然后是去饱和或羟基化；鞘氨醇的酰化；和水解更复杂的鞘脂，如鞘磷脂。这项资助的重点是负责鞘基酰化的酶（称为神经酰胺合成酶），我们最近已经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），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共底物的高度选择性，这些基因产生（二氢）-神经酰胺的特定分子亚种。在这项资助中，我们将进一步表征这些以及其他哺乳动物LASS同源物，以确定它们在鞘脂合成中的作用。这些研究将采用多种方法：利用基因克隆来表达感兴趣的基因，利用RNAi来抑制感兴趣的基因；神经酰胺合成酶活性的生化表征荧光显微镜探索亚细胞定位；以及液相色谱、电喷雾电离串联质谱（MS/MS和MS/MS/MS）对神经酰胺亚种、复杂鞘脂和其他感兴趣的代谢物（例如鞘碱1-磷酸盐等）进行深入、定量分析。质谱分析也将使用稳定同位素标记的骨干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）添加外源性鞘脂和类似物（包括药物safingol和FTY720）的情况下，特定LASS家族成员的mRNA表达水平如何调节神经酰胺的生物合成。这些研究将提供关于鞘脂代谢的基本信息，所选择的实验系统将提供神经酰胺合成酶在诸如癌症等疾病中的作用，其中已知存在LASS1表达异常，以及神经酰胺合成酶在鞘脂基治疗中的作用。

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.