Molecular characterization of lysosomal glucocerebrosidase

溶酶体是细胞内的一种小细胞器，对于保持我们的身体健康至关重要。溶酶体中包装并储存了十多种酶蛋白。它们的功能是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。不幸的是，溶酶体酶可能会因为基因突变而发生故障，干扰其产生或使其失活/不稳定。葡萄糖脑苷脂酶（GBA）就是这样的例子之一。 GBA 基因的有害突变会导致戈谢病，这是一种全民族最常见的溶酶体疾病。由于 GBA 缺乏，一种被称为葡萄糖脑苷脂的脂质在内脏器官、血液、骨骼、偶尔还有大脑的细胞中积累到非常高的有毒水平，导致神经变性和死亡。基因突变导致酶失去功能的机制尚不清楚。在本提案中，我们计划继续在DNA水平上鉴定GBA突变，并检查它们对GBA合成、结构/功能以及GBA向溶酶体转运的影响。由于戈谢病的突变也可能是由我们基因组中 2 个 GBA 基因拷贝之间的相互作用引起的（所有非灵长类哺乳动物只有 1 个拷贝），因此我们将调查其他灵长类动物中是否存在 2 个 GBA 拷贝，以便更多地了解反映我们过去的分子进化，以及这两个 GBA 基因如何在可能破坏 GBA 功能的“基因转换”和“重组”过程中相互作用。利用重组 DNA 和生物反应器/发酵技术，我们还将尝试在转基因昆虫和酵母细胞中生产大量正常人类 GBA，以供进一步研究。所生产的一种形式的 GBA 将具有额外的“蛋白质转导结构域”，并在实验小鼠中测试其穿越血脑屏障以治疗脑部疾病的能力。这项对 GBA 基因的研究可能有助于我们更好地了解人类是如何从其他灵长类动物进化而来的，以及该酶在正常和疾病条件下如何发挥作用，并制定治疗和预防策略。

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 interfere with its production or 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, bones, and occasionally the brain, causing neurodegeneration 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 bioreactor/fermentation technologies, we will also attempt to produce copious amounts of normal human GBA, in genetically-modified insect and yeast cells, for further studies. 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. This research on the GBA gene may help us to better understand how did human evolve from other primates and how does the enzyme function in normal and disease condition, and in devising strategies for treatment and prevention.