Endoplasmic reticulum quality control of mutant HexA enzyme in Tay-Sachs disease

描述（由申请人提供）：溶酶体储存障碍（lsd）是一类影响溶酶体-内体系统正常功能的毁灭性疾病。常见的结果是代谢中间体的隔离和随后的细胞死亡。在细胞周转率极低的中枢神经系统中，细胞成分的溶酶体加工尤为重要。因此，像Tay-Sachs病这样的lsd在一定程度上被定义为严重的神经退行性变，被诊断为lsd的儿童通常活不过5岁。泰-萨克斯病是由HEXA基因的单一突变引起的，这种突变导致酶-己糖氨酸酶A （HEXA）在到达溶酶体之前被降解。如果没有溶酶体定位的HexA， HexA的天然底物GM2神经节苷脂就不能正常降解，这些代谢物的积累导致细胞死亡。许多常见的HexA突变体从未离开内质网（ER），这是由于质量控制机制阻止酶在分泌途径中进行。内质网相关降解（ERAD）是一个组成过程，需要检测折叠错误的蛋白质并将其逆转录回细胞质，在那里它们被蛋白酶体降解。有趣的是，大多数HexA突变破坏了酶的折叠，但保留了活性位点的完整性。因此，改变质量

DESCRIPTION (provided by applicant): Lysosomal storage disorders (LSDs) are a devastating class of diseases which affect the ability of the lysosome-endosome system to properly function. The frequent outcome is sequestration of metabolic intermediates and subsequent cell death. Lysosomal processing of cellular components is particularly essential in the central nervous system, where cells have extremely low turnover rates. Therefore, LSDs such as Tay-Sachs disease are defined in part by severe neurodegeneration, with diagnosed children generally not surviving past the age of five. Tay-Sachs disease is caused by single mutations in the gene HEXA, which causes the enzyme ¿ -hexosaminidase A (HexA) to be degraded before it can reach the lysosome. The natural substrate for HexA, GM2 ganglioside, is not properly degraded without lysosome-localized HexA, and the buildup of these metabolites leads to cell death. It is well described that many common HexA mutants never exit the endoplasmic reticulum (ER) due to quality control mechanisms that prevent the enzyme from proceeding in the secretory pathway. ER-associated degradation (ERAD) is a constitutive process that entails the detection of malfolded proteins and their retrotranslocation back to the cytosol, where they are degraded by the proteasome. Interestingly, most HexA mutations disrupt the folding of the enzyme but leave the active site intact. Therefore, altering the quality control capabilities of the cell, either by allowing HexA more chances to fold, or by slowing its rate of degradation, should lead to an increase in proper localization and lysosomal activity of the enzyme. Indeed, studies with various LSDs have shown that even a small percent increase in activity at the lysosome may help prevent disease progression. The goal of this proposal is to study the detection and turnover of mutant HexA by ER factors. My hypothesis is that because of its biochemical properties, HexA will be triaged in a similar pathway used for other soluble glycoproteins, such as the ¿1- antitrypsin mutant, NHK. Using a combination of overexpression, RNA interference, and pharmacological agents, I will determine the temporal requirements for disposal of mutant HexA by chaperones and degradation machinery. Besides a targeted approach that will examine the roles of BiP, protein disulfide isomerases, and other important quality control factors, a SILAC-based proteomics approach will be used to uncover novel contributions of other ER-resident proteins. Using information about the mechanism of HexA degradation, I will specifically alter ER quality control to allow for increased lysosomal activityof mutant HexA. A deeper understanding of the turnover pathway of mutant HexA will allow for the use of drugs that can cross the blood- brain barrier and specifically alter the quality control factors important in the production of HexA without causing global ER stress or affecting general protein folding and ERAD.