ACID CERAMIDASE, CERAMIDE & FARBER DISEASE

描述(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)在鞘磷脂代谢、鞘磷脂介导的信号转导和Farber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)建立了大规模生产人酶的高效表达/纯化系统；c)对重组酶进行了广泛的鉴定，揭示了由AC、酸性鞘磷脂酶和至少另一种参与神经酰胺代谢的酶组成的多酶复合体；以及d)构建了第一个AC活性的敲除小鼠模型。在后两个实验中，在E8.5天或之后，没有发现纯合子、受影响的(Acko-/-)胚胎。我们现在建议通过追求以下四个具体目标来扩展这些发现：1)检测AC在小鼠早期发育中的表达，并探讨为什么没有Acko-/-胚胎的机制。我们将记录AC在正常小鼠胚胎中的表达模式，从Acko/-杂交中获得植入前胚胎进行基因分析和生化/形态鉴定，并研究Acko/-动物的配子，以揭示突变配子中可能影响受精的潜在异常。2)构建并鉴定AC条件性敲除小鼠。我们已经构建了AC基因打靶载体，可用于培育条件性KO小鼠。接下来，我们将获得这个靶向序列纯合的小鼠，并在可诱导的、巨噬细胞特异的和浦肯野细胞特异的启动子的控制下，将它们培育成表达Cre重组酶的转基因小鼠。由此产生的动物将在临床、病理和生化方面表现出特征。3)研究多酶复合体中AC、酸性鞘磷脂酶和其他脂类水解酶的相互作用。我们将使用新型的鞘磷脂亲和配体来获得大量的多酶复合体，并通过蛋白质组学方法和/或功能分析来鉴定新的成分，并研究在已知刺激鞘脂介导的细胞信号的细胞生长条件下复合体的形成和细胞内转运，以及4)使用AC特异性抑制剂来研究AC的正向和反向AC活性，以及用于Farber病的治疗。我们将继续在体外和在体上表征新的鞘磷脂类似物的抑制作用，并评估它们在Farber病伴侣疗法中的应用。

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.