Molecular characterization of lysosomal glucocerebrosidase

溶酶体葡萄糖脑苷脂酶的分子表征

• 批准号: 138216-2009
• 负责人: Choy, Francis
• 金额: $ 2.55万
• 依托单位: University of Victoria
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2010
• 资助国家: 加拿大
• 起止时间: 2010-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=451786
• 关键词: Molecular; characterization; lysosomal; glucocerebrosidase

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.

溶酶体是细胞内的一种小细胞器，对保持身体健康至关重要。十几种酶蛋白被包装并储存在溶酶体中。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，溶酶体酶可能由于基因突变而失效，使其失活/不稳定。其中一个例子就是葡萄糖脑苷酶（GBA）。GBA基因的有害突变导致戈谢病，这是一种泛种族和最常见的溶酶体疾病。由于GBA缺乏，一种被称为糖脑苷的脂质在内脏器官、血液和骨骼的细胞中积累到非常高的毒性水平，导致戈谢病，偶尔也会在大脑中，导致戈谢病的神经性致命形式和死亡。基因突变使酶丧失功能的机制尚不清楚。在本文中，我们计划继续在DNA水平上鉴定GBA突变，并研究它们对GBA合成、结构/功能和向溶酶体运输的影响。由于戈谢病的突变也可能是由于我们基因组中GBA基因的2个拷贝之间的相互作用造成的（所有非灵长类哺乳动物都只有1个拷贝），我们将调查其他灵长类动物中2个GBA拷贝的存在，以了解更多反映我们过去的分子进化，以及两个GBA基因如何在称为“基因转换”和“重组”的过程中相互作用，这可能会破坏GBA功能。利用重组DNA和干细胞技术，我们计划对神经性戈谢病小鼠模型（我们已经创建了非神经性小鼠模型）进行基因工程，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类GBA，用于进一步的生化分析。所产生的一种GBA将具有额外的“蛋白质转导结构域”，并将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。