Molecular characterization of lysosomal glucocerebrosidase

点击翻译按钮获取中文摘要

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.