ACID CERAMIDASE, CERAMIDE & FARBER DISEASE

描述（由申请人提供）：我们研究的总体目标是研究酸性神经酰胺酶（AC）在鞘脂代谢、鞘脂介导的信号转导以及法伯病的发病机制中的作用。为此，我们：a) 分离了编码人和鼠 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参与神经酰胺代谢的其他酶组成的多酶复合物，d) 构建了第一个 AC 活性的敲除小鼠模型。在后面的这些实验中，在 E8.5 天或之后没有发现纯合受影响的 (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）检查早期小鼠发育中 AC 的表达并研究解释 ACKO-/- 胚胎缺失的机制。我们将记录正常小鼠胚胎中 AC 的表达模式，从 ACKO /- 杂交获得植入前胚胎进行基因型分析和生化/形态学表征，并研究来自 ACKO /- 动物的配子，以揭示可能影响受精的突变配子中的潜在异常，2）构建和表征 AC 条件敲除小鼠。我们已经构建了AC基因打靶载体，可用于产生条件KO小鼠。接下来我们将获得该靶向序列纯合的小鼠，并将它们培育成在诱导型、巨噬细胞特异性和浦肯野细胞特异性启动子控制下表达Cre重组酶的转基因小鼠。所得动物将在临床、病理学和生化方面进行表征。 3) 研究多酶复合物中 AC、酸性鞘磷脂酶和其他脂质水解酶的相互作用。我们将使用新颖的鞘脂亲和配体来获得大量的多酶复合物，并通过蛋白质组学方法和/或功能测定来鉴定新的成分，并在已知刺激鞘脂介导的细胞信号传导的细胞生长条件下研究复合物的形成和细胞内运输，以及4)使用AC特异性抑制剂来研究“正向”和“反向”AC活性，并用于治疗法伯病。我们将继续表征新型鞘脂类似物的体外和原位抑制作用，并评估它们在法伯病伴侣治疗中的用途。

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.