CERAMIDASES, CERAMIDE, AND FARBER DISEASE

神经酰胺在鞘磷脂代谢中起关键作用，是神经节苷脂、髓鞘成分(如硫脂)和膜成分(如鞘磷脂和复合糖脂)合成和降解的中间产物。神经酰胺及其分解代谢产物鞘氨醇是重要的细胞信号分子，参与细胞程序性死亡和/或细胞生长。已在哺乳动物细胞中鉴定出几种神经酰胺酶，其中一种酶-酸性神经酰胺酶(AC)的缺失会导致遗传疾病Farber病。这项建议的总体目标是研究神经酰胺酶和基因家族的生物学，因为它与鞘磷脂代谢、Farber病病理生物学和信号转导有关。本实验室的相关初步数据包括：a)分离了编码人和小鼠AC的全长cDNA和基因组序列；b)建立了基于荧光的体外和原位神经酰胺酶活性检测系统，分析了神经酰胺酶在小鼠组织中的表达，并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表达AC；c)分离了几种AC相关蛋白的cDNA克隆，并对这些新序列的功能进行了初步研究；d)鉴定了导致Farber病的AC基因的第一个突变；以及e)构建了AC基因敲除小鼠。这项拨款申请的具体目的是：1)继续研究小鼠组织中神经酰胺酶的活性和RNA表达，特别关注潜在的发育、组织特异性和应激相关的调控；2)建立稳定过表达人AC的哺乳动物细胞系，纯化过表达的酶，并通过定点突变和新的酶分析方法研究其结构/功能特性；3)研究AC基因敲除小鼠与Farber病和鞘磷脂介导的信号转导有关的病理生物学特征；以及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研究提供重要的见解，并为研究神经酰胺和鞘氨醇在信号转导和Farber病的病理生理中的作用提供必要的试剂。

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.