Protein Misprocessing in Krabbe Disease

克拉伯病中的蛋白质加工错误

• 批准号: 7124133
• 负责人: CHRISTOPHER B ECKMAN
• 金额: $ 19万
• 依托单位: MAYO CLINIC JACKSONVILLE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7124133
• 关键词: Krabbe' s disease; cell line; chemotherapy; colorimetry; drug discovery /isolation; drug screening /evaluation; enzyme activity; enzyme inhibitors; enzyme structure; enzyme substrate; gene mutation; hydrolase; protein folding; protein localization; protein structure function; protein transport; recombinant proteins; small molecule

DESCRIPTION (provided by applicant): Globoid cell Leukodystrophy (GLD; Krabbe disease), is a devastating lysosomal storage disorder caused by mutations in galactosylceramidase (GALC) that severely impair enzymatic activity. Affected individuals typically present with symptoms in the first few months of life. Disease progression is generally rapid, leading to death within 1-2 years. Current treatment options for the disease are very limited. At least 62 mutations in the GALC gene have been identified that cause disease. The majority of these mutations are missense mutations that lie outside of the catalytic domain. We have recently demonstrated that at least several of these mutations appear to affect enzymatic activity by causing the enzyme to be misprocessed and subsequently degraded, similar to other misfolded protein disorders such as nephrogenic diabetes insipidus, primary hyperoxaluria type 1, congenital nephrotic syndrome, and other lysosomal storage diseases such as Gaucher disease and Fabry disease. In many of these instances small molecular weight inhibitors of the affected proteins themselves can "trick" the quality control machinery of the cell to recognize the mutant protein as normal thus allowing it reach the appropriate organelle where it becomes active. Using this approach we have already screened through a small library of compounds and identified one inhibitor that when applied to cells harboring a mutant form of GALC resulted in a substantial increase in GALC enzymatic activity. In this exploratory application we propose to expand our in vitro screens to identify additional inhibitors of GALC and determine whether or not these compounds can influence mutant GALC enzymatic activity in model systems. Further we propose to determine the subcellular localization of this increased activity to determine if the enzyme has reached its proper location and to monitor natural substrate cleavage as a prelude to in vivo testing.

描述（由申请人提供）：球形细胞白细胞营养不良（GLD； Krabbe病）是由半乳糖酶酶（GALC）引起的毁灭性溶酶体储存障碍，严重损害了酶促活性。受影响的个体通常在生命的头几个月出现症状。疾病进展通常很快，在1 - 2年内导致死亡。该疾病的当前治疗选择非常有限。已经确定了引起疾病的GALC基因中至少有62个突变。这些突变中的大多数是位于催化域之外的错义突变。 We have recently demonstrated that at least several of these mutations appear to affect enzymatic activity by causing the enzyme to be misprocessed and subsequently degraded, similar to other misfolded protein disorders such as nephrogenic diabetes insipidus, primary hyperoxaluria type 1, congenital nephrotic syndrome, and other lysosomal storage diseases such as Gaucher disease and Fabry disease.在许多情况下，受影响蛋白质本身的小分子量抑制剂可以“欺骗”细胞的质量控制机制以识别突变蛋白正常，从而使其到达其活性的适当细胞器。使用这种方法，我们已经通过一个小的化合物库进行筛选，并确定了一种抑制剂，当应用于具有突变形式的GALC的细胞时，导致GALC酶促活性大幅增加。在此探索性应用中，我们建议扩展我们的体外筛选，以鉴定加尔克的其他抑制剂，并确定这些化合物是否会影响模型系统中突变的GALC酶活性。此外，我们建议确定这种增加活性的亚细胞定位，以确定该酶是否已达到其适当位置并监测自然底物裂解，以作为体内测试的前奏。