CERAMIDASES, CERAMIDE, AND FARBER DISEASE

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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.