Molecular characterization of lysosomal glucocerebrosidase

溶酶体葡萄糖脑苷脂酶的分子表征

• 批准号: 138216-2006
• 负责人: Choy, Francis
• 金额: $ 2.04万
• 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: 2008
• 资助国家: 加拿大
• 起止时间: 2008-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=391461
• 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 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.

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，溶酶体酶可能因为干扰其产生或使其失活/不稳定的基因突变而发生故障。一个这样的例子是葡糖脑苷脂酶（GBA）。GBA基因的有害突变导致戈谢病，一种泛种族和最常见的溶酶体疾病。由于GBA缺乏，一种称为葡萄糖脑苷脂的脂质在内脏器官，血液，骨骼，偶尔大脑的细胞中积累到非常高的毒性水平，导致神经变性和死亡。基因突变使酶失去功能的机制还不是很清楚。在这个提议中，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/功能和转运到溶酶体的影响。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引起的，（所有非灵长类哺乳动物只有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，以了解更多关于其反映我们过去的分子进化，以及两个GBA基因如何在被称为“基因转换”和“重组”的过程中相互作用，从而可能破坏GBA的功能。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的正常人GBA，以供进一步研究。产生的一种形式的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过血脑屏障治疗脑部疾病的能力。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，以及这种酶在正常和疾病状态下如何发挥作用，以及如何制定治疗和预防策略。