ACID CERAMIDASE, CERAMIDE & FARBER DISEASE

描述（由申请人提供）：我们研究的总体目标是探讨酸性神经酰胺酶（AC）在鞘脂代谢、鞘脂介导的信号转导和法伯病发病机制中的作用。为此，我们已经：a)分离了编码人和小鼠AC的全长cdna和基因，b)开发了用于大规模生产人类酶的过表达/纯化系统，c)广泛表征了重组酶，揭示了由AC，酸性鞘磷脂酶和至少一种其他参与神经酰胺代谢的酶组成的多酶复合物，d)构建了第一个敲除AC活性的小鼠模型。在这些实验中，没有发现纯合子，影响（ACKO-/-）胚胎在E8.5天或以后。我们现在建议通过以下四个具体目标来扩展这些发现：1)检查AC在小鼠早期发育中的表达，并研究ACKO-/-胚胎缺失的机制。我们将记录AC在正常小鼠胚胎中的表达模式，从ACKO+/-杂交获得着床前胚胎进行基因型分析和生化/形态学表征，并研究ACKO+/-动物的配子，以发现可能影响受精的突变配子的潜在异常。2)构建和表征AC条件敲除小鼠。我们已经构建了一个AC基因靶向载体，可用于培养条件型KO小鼠。接下来，我们将获得针对该靶向序列的纯合子小鼠，并在诱导型、巨噬细胞特异性和浦肯野细胞特异性启动子的控制下，将它们培育成表达Cre重组酶的转基因小鼠。由此产生的动物将具有临床、病理和生物化学特征。3)研究AC、酸性鞘磷脂酶和其他脂质水解酶在多酶复合物中的相互作用。我们将使用新颖的鞘脂亲和配体获得大量的多酶复合物，并通过蛋白质组学方法和/或功能分析鉴定新的成分，并研究已知刺激鞘脂介导的细胞信号传导的细胞生长条件下复合物的形成和细胞内运输。4)使用AC特异性抑制剂研究“正向”和“反向”AC活性，并用于法伯病的治疗。我们将继续在体外和原位研究新型鞘脂类似物的抑制作用，并评估其在法伯病的伴侣治疗中的应用。

DESCRIPTION (provided by applicant): The overall goals of our research are to investigate the role of acid ceramidase (AC) in sphingolipid metabolism, sphingolipid-mediated signal transduction, and the pathogenesis of Farber disease. Towards this end, we have: a) isolated the full-length cDNAs and genes encoding human and murine AC, b) developed an overexpression/purification system for the large-scale production of the human enzyme, c) extensively characterized the recombinant enzyme, revealing a multienzyme complex consisting of AC, acid sphingomyelinase, and at least one other enzyme involved in ceramide metabolism, and d) constructed the first knock-out mouse model of AC activity. In these latter experiments, no homozygous, affected (ACKO-/-) embryos were found at day E8.5 or later. We now propose to extend these findings by pursuing the following four specific aims: 1) Examine the expression of AC in early mouse development and investigate the mechanism explaining the absence of ACKO-/- embryos. We will document the expression pattern of AC in normal mouse embryos, obtain preimplantation embryos from ACKO+/- intercrosses for genotype analysis and biochemical/morphological characterization, and study the gametes from ACKO+/- animals to uncover potential abnormalities in mutant gametes that might affect fertilization, 2) Construct and characterize AC conditional knock-out mice. We have already constructed an AC gene targeting vector that can be used to produce conditional KO mice. We will next obtain mice that are homozygous for this targeting sequence, and breed them to transgenic mice expressing Cre recombinase under the control of inducible, macrophage-specific, and Purkinje cell-specific promoters. Resulting animals will be characterized clinically, pathologically, and biochemically. 3) Investigate the interaction of AC, acid sphingomyelinase and other lipid hydrolases in a multienzyme complex. We will use novel, sphingolipid affinity ligands to obtain large quantities of the multienzyme complex, and identify new components by a proteomics approach and/or by functional assays, and study the formation and intracellular trafficking of the complex under cell growth conditions known to stimulate sphingolipid-mediated cell signaling, and 4) Use AC-specific inhibitors to investigate the "forward" and "reverse" AC activities, and for the treatment of Farber disease. We will continue to characterize the inhibitory effects of novel sphingolipid analogues in vitro and in situ, and evaluate their use for chaperone therapy of Farber disease.