Protein Misprocessing in Krabbe Disease

描述（由申请人提供）：Globoid cell Leukodystrophy （GLD； Krabbe病）是一种毁灭性的溶酶体储存疾病，由半乳糖神经酰胺酶（GALC）突变引起，严重损害酶活性。受影响的个体通常在生命的最初几个月出现症状。疾病进展通常很快，可在1-2年内导致死亡。目前针对该疾病的治疗方案非常有限。至少有62种GALC基因突变已被确定为致病基因。这些突变中的大多数是位于催化结构域之外的错义突变。我们最近已经证明，至少有几种突变似乎通过导致酶被错误加工并随后降解来影响酶的活性，类似于其他错误折叠的蛋白质疾病，如肾源性尿囊症、原发性高草酸尿症1型、先天性肾病综合征和其他溶酶体储存疾病，如戈谢病和法布里病。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，使其识别突变蛋白为正常蛋白，从而使其到达适当的细胞器，在那里它变得活跃。使用这种方法，我们已经筛选了一个小的化合物库，并确定了一种抑制剂，当应用于含有GALC突变形式的细胞时，会导致GALC酶活性大幅增加。在这个探索性应用中，我们建议扩大我们的体外筛选，以确定GALC的其他抑制剂，并确定这些化合物是否可以影响模型系统中突变GALC的酶活性。此外，我们建议确定这种增加的活性的亚细胞定位，以确定酶是否已经达到其适当的位置，并监测自然底物切割作为体内测试的前奏。

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.