LIPASE AND CATHEPSIN ABNORALITIES IN BATTEN DISEASE

这是一个应用程序的更新，其广泛的长期目标是 了解巴顿病的遗传和生化基础 （神经元蜡样灰褐质沉着症; NCL）。 将特别强调 了解晚期婴儿（NCL 2）和青少年（NCL 3）的形式， 因为这些是遗传性失明、癫痫发作的常见形式 我们将测试NCL的假设，即NCL的结果来自于 不能运输和降解正常的线粒体蛋白质（ 线粒体ATP合酶的蛋白脂亚基c（c- 简称肽）。这种肽的溶酶体储存，主要是 在神经元、视网膜色素上皮细胞和胰腺中， 假设引起许多溶酶体常见临床症状 贮藏病害我们将从NCL组织中纯化C肽， 用[14 C]DCCD或[125 I]标记，用特异性多克隆抗体鉴定， 检查任何异常的翻译后修饰，如三甲基- 赖氨酸残基，并在加载正常和NCL后跟踪其催化剂 成纤维细胞和淋巴母细胞样细胞系。这只会间接地 因此，我们通过PCR获得了绵羊C肽的cDNA， 含有巨细胞病毒启动子等的质粒载体， 在神经母细胞瘤细胞系F-11中过表达该基因。这些和相关 线是CNS的模型。我们还将检查正常和NCL 淋巴母细胞样细胞系。 我们将尝试创造自发荧光 色素储存，然后表明，这也导致长萜醇糖 积累，溶酶体磷脂酶A1抑制。和其他生化 NCL的特征。我们将确定泛素途径是否 通常参与c肽的处理，寻找c肽的转运 蛋白质通过c-肽亲和层析的脑提取物和外观 对于脂蛋白复合物中的C-肽， 氯化铯梯度范围为1.063- 1.21 g/ml 超浓缩的任何突变都将在大量的细胞中进行检测。 可从患者获得的品系，专性杂合子（正常的50 活动）和可靠的诊断测试将被设计。理解 NCL中的缺陷显然将为细胞生物学开辟一个全新的领域 处理疏水肽的运输和处理。 最后， 探测航母的能力将在作战中具有巨大的价值 这种儿童神经退行性疾病

This is a renewal of an application whose broad, long-term objectives are to understand the genetic and biochemical basis of Batten disease (Neuronal Ceroid Liofuscinosis; NCL). Particular emphasis will be placed on understanding the late-infantile (NCL2) and juvenile (NCL3) forms of the disease since these are common forms of inherited blindness, seizures and dementia in the U.S. We will test the hypothesis that NCL results from the inability to transport and degrade a normal mitochondrial protein (the DCCD-binding, proteolipid subunit c of mitochondrial ATP synthase (c- peptide for short). The lysosomal storage of this peptide, predominantly in neurons, retinal pigmentary epithelial cells and pancreas, is then hypothesized to give rise to clinical symptoms common to many lysosomal storage diseases. We will purify the c-peptide from NCL tissue, label it with [14C]DCCD or [125I], identify it by specific polyclonal antibody, check for any abnormal post-translational modifications such as trimethyl- lysine residues, and follow its catabolism after loading of normal and NCL fibroblasts and lymphoblastoid cell lines. This will only give indirect evidence so we have obtained cDNA for sheep c-peptide by PCR, spliced into a plasmid vector which contains a cytomegalovirus promoter etc. and stably overexpressed the gene in neuroblastoma cell line F-11. These and related lines are a model for the CNS. We will also transfect both normal and NCL lymphoblastoid cell lines. We will attempt to create autofluorescent pigment storage and then show that this also results in dolichol sugar accumulation, lysosomal phospholipase A1 inhibition.and other biochemical characteristics of NCL. We will determine if the ubiquitin pathway is normally involved in c-peptide disposal, look for a c-peptide transport protein by c-peptide affinity chromatography of brain extracts and look for c-peptide in lipoprotein complexes by their flotation densities in the range of 1.063-1.21g/ml on cesium chloride gradients in the ultracentrifuge. Any mutation will be assayed in the large number of cell lines available from patients, obligate heterozygotes (50% of normal activity) and a reliable diagnostic test will be devised. Understanding the defect in NCL will clearly open up a, whole new area of cell biology dealing with the transport and disposal of hydrophobic peptides. Finally, the, ability to detect carriers will have tremendous value in combatting this devastating neurodegenerative disorder of children.