ACID CERAMIDASE, CERAMIDE & FARBER DISEASE

酸性神经酰胺酶、神经酰胺

基本信息

批准号：
7992518
负责人：
EDWARD H. SCHUCHMAN
金额：
$ 2.5万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-01 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7992518
关键词：
Affect Affinity Affinity Chromatography Animals Biochemical Biological Assay Breeding Cell Survival Cells Ceramides Chinese Hamster Ovary Cell Complex Development Embryo Embryonic Development Enzymes Farber&apos s lipogranulomatosis Fertilization Gene Proteins Gene Targeting Genes Genotype Germ Cells Goals Growth Human Hydrolase Immunofluorescence Immunologic In Situ In Situ Hybridization In Vitro Individual Knock-out Knockout Mice Lead Length Ligands Lipids Mediating Metabolism Molecular Chaperones Multienzyme Complexes Mus Partner in relationship Pathogenesis Patients Pattern Phorbol Esters Physiological Property Proteomics Purkinje Cells RNA Reagent Recombinants Research Role Signal Transduction Sphingolipids System Transgenic Mice Western Blotting acid sphingomyelinase analog animal breeding blastocyst cell growth embryonic stem cell galactosylgalactosylglucosylceramidase inhibitor/antagonist irradiation large scale production macrophage mouse development mouse model mutant novel overexpression polypeptide promoter recombinase research study trafficking vector

项目摘要

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.

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和Farber疾病的发病机理中的作用。为此，我们有：a）隔离了编码人和鼠类AC的全长cDNA和基因，b）开发了一种过表达/纯化系统，用于大规模生产人类酶，c）广泛地表征了重组酶，即揭示了由AC，酸鞘磷脂酶和至少一种参与神经酰胺代谢的酶组成的多烯酶复合物，d）构建了第一个AC活性的敲除小鼠模型。在这些后一个实验中，在E8.5天或以后没有发现纯合子（ACKO - / - ）胚胎。现在，我们建议通过追求以下四个特定目的来扩展这些发现：1）检查早期小鼠发育中AC的表达，并研究解释ACKO - / - 胚胎的机制。我们将记录AC在正常小鼠胚胎中的表达模式，从ACKO +/- - 间交叉获得基因型分析和生化/形态特征的植入前胚胎，并研究从ACKO +/-动物到可能影响肥料的潜在异常的配子，2）构造和表征AC条件敲除小鼠。我们已经构建了一个可用于产生条件KO小鼠的AC基因靶向载体。接下来，我们将获得该靶向序列纯合的小鼠，并将其培养为在诱导，巨噬细胞特异性和Purkinje细胞特异性启动子的控制下表达CRE重组酶的转基因小鼠。由此产生的动物将在临床，病理和生化上进行表征。 3）研究多酶复合物中AC，酸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力配体来获得大量的多烯酶复合物，并通过蛋白质组学方法和/或通过功能测定来识别新成分，并研究复合物在已知刺激鞘脂的细胞生长条件下复合物的形成和细胞内运输 - 介导的细胞信号传导，以及4）使用AC特异性抑制剂研究“正向”和“反向” AC活性，以及治疗Farber疾病。我们将继续表征在体外和原位新型鞘脂类似物的抑制作用，并评估它们用于伴侣治疗法伯疾病的抑制作用。