CERAMIDASES, CERAMIDE, AND FARBER DISEASE

神经酰胺酶、神经酰胺和法伯病

• 批准号: 6489720
• 负责人: EDWARD H. SCHUCHMAN
• 金额: $ 30.78万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-15 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6489720
• 关键词: Adenoviridae; Retroviridae; amidohydrolases; ceramides; complementary DNA; enzyme activity; enzyme induction /repression; genetically modified animals; laboratory mouse; lipogranulomatosis; nucleic acid sequence; phosphodiesterases; site directed mutagenesis; sphingosine; tissue /cell culture; transfection /expression vector

Ceramide plays a key role in sphingolipid metabolism, serving as an intermediate for the synthesis and degradation of gangliosides, myelin constituents, such as sulfatides, and membrane components, such as sphingomyelin and complex glycolipids. It also has been suggested that ceramide and its catabolic product, sphingosine, are important cell signaling molecules involved in programmed cell death and/or cell growth. Several ceramidases have been identified in mammalian cells, and a deficiency of one of these enzymes, acid ceramidase (AC), leads to the genetic disorder, Farber disease. The overall goal of this proposal is to investigate the biology of the ceramidase enzyme and gene family as it relates to sphingolipid metabolism, Farber disease pathobiology, and signal transduction.. Pertinent preliminary data from our laboratory includes the: a) isolation of full-length cDNA and genomic sequences encoding human and murine AC; b) development of fluorescence-based assay systems to assess ceramidase activities in vitro and in situ, analysis of ceramidase expression in mouse tissues, and over-expression of AC in mammalian cells using adenoviral and retroviral gene transfer vectors; c) isolation of cDNA clones encoding several AC-related proteins, and initial investigation of the function of these novel sequences; d) identification of the first mutation in the AC gene causing Farber disease; and e) construction of an AC knock-out mouse. The specific aims of this grant application are to: 1) continue investigating ceramidase activity and RNA expression in mouse tissues, paying particular attention to potential developmental, tissue-specific, and stress-related regulation; 2) develop a mammalian cell line stably over-expressing human AC, purify the over- expressed enzyme, and investigate its structure/function properties by site-directed mutagenesis and novel enzyme assay procedures; 3) characterize the pathobiology of the AC knock-out mouse as it relates to Farber disease and sphingolipid-mediated signal transduction; and 4) continue to investigate the biology of the cloned ceramidase-related sequences, including the development of expression vectors that will permit assessment of intracellular localization and potential catalytic functions. We believe that these studies will provide important insights into the biology of this important enzyme system, as well as essential reagents to study the roles of ceramide and sphingosine in signal transduction and the pathophysiology of Farber disease.

神经酰胺在鞘脂代谢中发挥着关键作用，作为神经节苷脂、髓磷脂成分（如硫苷脂）和膜成分（如鞘磷脂和复合糖脂）合成和降解的中间体。还有人提出，神经酰胺及其分解代谢产物鞘氨醇是参与程序性细胞死亡和/或细胞生长的重要细胞信号分子。哺乳动物细胞中已鉴定出多种神经酰胺酶，其中一种酶——酸性神经酰胺酶（AC）的缺乏会导致遗传性疾病——法伯病。该提案的总体目标是研究神经酰胺酶和基因家族的生物学，因为它与鞘脂代谢、法伯病病理学和信号转导有关。来自我们实验室的相关初步数据包括：a）全长分离编码人和鼠AC的cDNA和基因组序列； b) 开发基于荧光的测定系统，以评估神经酰胺酶的体外和原位活性，分析小鼠组织中的神经酰胺酶表达，以及使用腺病毒和逆转录病毒基因转移载体在哺乳动物细胞中过度表达AC； c) 分离编码几种AC相关蛋白的cDNA克隆，并对这些新序列的功能进行初步研究； d) 鉴定导致法伯病的 AC 基因中的第一个突变； e) AC 敲除小鼠的构建。本次资助申请的具体目的是：1）继续研究小鼠组织中的神经酰胺酶活性和RNA表达，特别关注潜在的发育、组织特异性和应激相关的调节； 2）开发稳定过表达人AC的哺乳动物细胞系，纯化过表达的酶，并通过定点诱变和新的酶测定方法研究其结构/功能特性； 3) 表征 AC 敲除小鼠的病理学特征，因为它与法伯病和鞘脂介导的信号转导有关； 4) 继续研究克隆的神经酰胺酶相关序列的生物学，包括开发能够评估细胞内定位和潜在催化功能的表达载体。我们相信，这些研究将为这一重要酶系统的生物学提供重要的见解，并为研究神经酰胺和鞘氨醇在信号转导和法伯病的病理生理学中的作用提供重要的试剂。