Characterization of mammalian ceramide synthases

哺乳动物神经酰胺合酶的表征

基本信息

批准号：
7414590
负责人：
ALFRED Harrison MERRILL
金额：
$ 28.51万
依托单位：
GEORGIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7414590
关键词：
Acyl Coenzyme A Acylation Affect Anabolism Antibodies Apoptosis Atherosclerosis Biochemical Biological Biological Assay Cell Proliferation Regulation Cell membrane Cell physiology Cells Ceramides Chemotherapy-Oncologic Procedure Code Complex Condition Depth Development Disease Electrospray Ionization Enzymes Facility Construction Funding Category Family member Fluorescence Microscopy Gene Expression Gene Family Genes Glycolipids Goals Grant Homologous Gene Homologous Protein Human Hybrids Hydrolysis Hydroxylation In Vitro Investigation Ions Liquid Chromatography Longevity Malignant Neoplasms Mammalian Cell Metabolism Methods Modification Molecular Mus Numbers Pathway interactions Peptides Pharmaceutical Preparations Play Process Production Property Protein Overexpression Proteins Purpose RNA Interference Range Regulation Role Safingol Small Interfering RNA Specific qualifier value Sphingolipids Sphingomyelins Sphingosine Stable Isotope Labeling Structure-Activity Relationship System Therapeutic Vertebral column analog base dihydroceramide dihydroceramide desaturase disorder control enzyme substrate gene cloning in vivo inorganic phosphate insight instrument interest mRNA Expression novel research study senescence serine palmitoyltransferase sphinganine stearoyl-coenzyme A tandem mass spectrometry tool

项目摘要

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.

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成分，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：二氢鞘氨醇酰化为二氢神经酰胺，然后去饱和或羟基化；鞘氨醇的酰化；以及更复杂的鞘脂的水解，例如鞘磷脂。这笔资助重点关注负责鞘氨醇碱基酰化的酶（称为神经酰胺合酶），我们最近为此确定了三个哺乳动物基因（称为 LASS1、4 和 5，表示长寿保证基因同源物），每个基因编码（二氢）神经酰胺合酶，产生由于对脂肪酰辅酶A辅助底物具有高度选择性，因此形成了（二氢）-神经酰胺的特定分子亚种。在这笔资助中，我们将进一步表征这些以及其他哺乳动物 LASS 同系物，以确定各自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感兴趣的基因，并使用 RNAi 来抑制感兴趣的基因；神经酰胺合酶活性的生化特征；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），用于对神经酰胺亚种、复杂鞘脂和其他感兴趣的代谢物（例如鞘氨醇碱 1-磷酸盐等）进行深入定量分析。质谱分析还将用于使用稳定同位素标记的主链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）通过添加外源鞘脂和类似物（包括药物 safingol 和 FTY720。这些研究将提供有关鞘脂代谢的基本信息，选定的实验系统将深入了解神经酰胺合酶在癌症等疾病中的作用，其中已知 LASS1 表达异常，以及神经酰胺合酶参与神经酰胺合酶的参与。基于鞘脂的疗法的作用。