Molecular characterization of lysosomal glucocerebrosidase

溶酶体是细胞内的一个小细胞器，对保持我们的身体健康至关重要。十几种酶蛋白被包装并储存在溶酶体内。它们通过消化和清除有害的代谢产物以及细菌和病毒等外来入侵者来发挥作用。不幸的是，溶酶体酶可能会因为基因突变而失灵，使其失去活性/不稳定。一个这样的例子是葡萄糖脑苷酶(GBA)。GBA基因的有害突变会导致高谢病，这是一种泛种族和最常见的溶酶体疾病。由于GBA缺乏，一种名为葡萄糖脑苷的脂质在内脏器官、血液和骨骼的细胞中积聚到非常高的有毒水平，导致高谢病，有时还会在大脑中，导致神经病理的致命形式的高谢病和死亡。基因突变使酶失去功能的机制(S)还不是很清楚。在这项建议中，我们计划继续在DNA水平上鉴定GBA突变，并检测它们对GBA的合成、结构/功能和运输到溶酶体的影响。由于高谢病的突变也可能是基因组中GBA基因的两个拷贝之间相互作用的结果(所有非灵长类哺乳动物只有一个拷贝)，我们将调查其他灵长类动物中是否存在这两个GBA拷贝，以便更多地了解它反映我们过去的分子进化，以及这两个GBA基因是如何在被称为“基因转换”和“重组”的过程中相互作用的，这可能会破坏GBA的功能。利用重组DNA和干细胞技术，我们计划从基因上设计一个神经性高谢病小鼠模型(我们已经创建了一个非神经性高谢病小鼠模型)，以研究大脑中的细胞生物学和病理生理学。我们还将尝试在转基因昆虫或酵母细胞中生产大量的人GBA，用于进一步的生化分析。一种产生的GBA将有一个额外的“蛋白质转导结构域”，并在实验小鼠身上测试其跨越血脑屏障治疗脑部疾病的能力。

The lysosome is a small organelle within the cell essential for keeping our body healthy. More than a dozen of enzyme-proteins are packaged and stored within the lysosome. They function by digesting and removing harmful metabolic products as well as foreign invaders such as bacteria and viruses. Unfortunately, a lysosomal enzyme may malfunction because of genetic mutations that render it inactive/unstable. One such example is the enzyme glucocerebrosidase (GBA). Harmful mutations in the GBA gene result in Gaucher disease, a panethnic and most frequent lysosomal disorder. Because of GBA deficiency, a lipid known as glucocerebroside accumulates to very high and toxic levels in the cells of visceral organs, blood, and bones that results in Gaucher disease, and occasionally in the brain, resulting in the neuropathic, lethal form of Gaucher disease and death. The mechanism(s) by which the genetic mutations render the enzyme nonfunctional is not very well understood. In this proposal, we plan to continue the identification of the GBA mutations at the DNA level and examine their effects on the synthesis, structure/function, and transport of GBA to the lysosome. Since mutations in Gaucher disease may also result from interaction between the 2 copies of the GBA gene in our genome (all non-primate mammals have only 1 copy), we will survey the presence of the 2 GBA copies in other primates in order to learn more about its molecular evolution which reflects our past, and how the two GBA genes interact in processes known as `gene conversion' and `recombination' that may disrupt GBA function. Using recombinant DNA and stem cell technologies, we plan to genetically engineer a mouse model of neuropathic Gaucher disease (we already have created a non-neuropathic mouse model) to study the cell biology and pathophysiology in the brain. We will also attempt to produce copious amounts of human GBA, in genetically-modified insect or yeast cells, for further biochemical analysis. One form of GBA produced will have an extra `protein transduction domain' and be tested in experimental mice for its ability to cross the blood-brain barrier for treatment of brain disease.