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)是一种破坏性的疾病，会影响溶酶体-内体系统正常运作的能力。常见的结果是代谢中间产物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，溶酶体对细胞成分的处理尤其重要。因此，像泰-萨克斯病这样的LSD在一定程度上被定义为严重的神经退化，确诊的儿童通常活不过五岁。泰-萨克斯病是由基因HEXA的单一突变引起的，该基因导致己糖氨酸酶A(HEXA)在到达溶酶体之前被降解。神经节苷脂GM2的天然底物不能在没有溶酶体定位的情况下被适当降解，这些代谢物的积聚会导致细胞死亡。众所周知，许多常见的六角体突变体永远不会离开内质网(ER)，这是因为质量控制机制阻止了酶在分泌途径中进行。内质网相关降解(ERAD)是一个结构性过程，它需要检测错误折叠的蛋白质并将它们反向移位到胞浆，在那里它们被蛋白酶体降解。有趣的是，大多数六角体突变会破坏酶的折叠，但保持活性部位不变。因此，改变质量 细胞的控制能力，无论是通过允许六六六有更多的机会折叠，还是通过减缓其降解速度，都应该导致酶的适当定位和溶酶体活性的增加。事实上，对不同LSD的研究表明，即使溶酶体活性略有增加，也可能有助于防止疾病的进展。本方案的目的是研究ER因子对突变型HexA的检测和周转。我的假设是，由于其生化特性，六六六将以类似于其他可溶性糖蛋白的途径进行筛选，例如1-抗胰蛋白酶突变体NHK。利用过表达、RNA干扰和药理作用的组合，我将确定伴侣和降解机械处理突变的六六A的时间要求。除了研究BiP、蛋白质二硫键异构酶和其他重要质量控制因素的作用外，还将使用基于SILAC的蛋白质组学方法来发现其他内质网驻留蛋白质的新贡献。利用HexA降解机制的信息，我将专门改变ER质量控制，以允许突变的HexA的溶酶体活性增加。对突变型HexA周转途径的深入了解将使药物的使用能够跨越血脑屏障，并特异性地改变在生产HexA过程中重要的质量控制因素，而不会导致全球内质网应激或影响一般的蛋白质折叠和ERAD。

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.