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

泰-萨克斯病突变型 HexA 酶的内质网质量控制

• 批准号: 8593531
• 负责人: Devin Dersh
• 金额: $ 2.87万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8593531
• 关键词: Active Sites; Affect; Age; Alleles; Ashkenazim; Back; Binding; Biochemical; Biochemical Process; Biological Assay; Birth; Blindness; Blood - brain barrier anatomy; Cell Death; Cell physiology; Cells; Child; Chronic; Classification; Cytosol; Data; Degradation Pathway; Detection; Diagnosis; Disease; Disease Progression; Drug usage; Endoplasmic Reticulum; Endosomes; Enzymes; Family; G(M2) Ganglioside; Gangliosidoses; Gene Mutation; Genes; Glycoproteins; Goals; Half-Life; Hex A; Hong Kong; Label; Lead; Left; Longevity; Lysosomes; Measures; Metabolic; Molecular Chaperones; Mutation; Nerve Degeneration; Neuraxis; Neurons; Outcome; Outcomes Research; Oxidation-Reduction; Paralysed; Pathway interactions; Patients; Pharmaceutical Preparations; Physiologic pulse; Play; Polysaccharides; Population; Process; Production; Property; Protein C Inhibitor; Protein Disulfide Isomerase; Proteins; Proteomics; Quality Control; RNA Interference; Role; Seizures; System; Tay-Sachs Disease; Techniques; Testing; Translating; Triage; Work; base; crosslink; deafness; design; disease phenotype; endoplasmic reticulum stress; enzyme activity; follower of religion Jewish; infancy; multicatalytic endopeptidase complex; mutant; novel; overexpression; prevent; protein folding; public health relevance; small hairpin RNA

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.

描述（由申请方提供）：溶酶体贮积症（LSD）是一类破坏性疾病，影响溶酶体-内体系统正常发挥功能的能力。常见的结果是代谢中间产物的螯合和随后的细胞死亡。细胞组分的溶酶体加工在中枢神经系统中特别重要，其中细胞具有极低的周转率。因此，像泰-萨二氏病这样的LSD在一定程度上被定义为严重的神经变性，被诊断出的儿童通常不能存活超过5岁。泰-萨二氏病是由HEXA基因的单一突变引起的，这导致酶己糖胺酶A（HexA）在到达溶酶体之前被降解。HexA的天然底物GM 2神经节苷脂在没有溶酶体定位的HexA的情况下不能适当降解，并且这些代谢物的积累导致细胞死亡。许多常见的HexA突变体从未退出内质网（ER），这是由于质量控制机制，阻止酶在分泌途径中进行。ER相关降解（ERAD）是一个组成性过程，需要检测错误折叠的蛋白质及其回转位回胞质溶胶，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，但保留了完整的活性位点。因此，改变质量 通过允许HexA有更多的机会折叠或通过减缓其降解速率，细胞的控制能力应导致酶的适当定位和溶酶体活性的增加。事实上，对各种LSD的研究表明，即使溶酶体活性增加一小部分，也可能有助于防止疾病进展。本研究的目的是研究ER因子对突变型HexA的检测和周转。我的假设是，由于它的生化特性，HexA将在用于其他可溶性糖蛋白的类似途径中被分类，例如1-抗胰蛋白酶突变体NHK。使用过表达，RNA干扰和药理学试剂的组合，我将确定的时间要求处理突变HexA的伴侣和降解机械。除了有针对性的方法，将检查BiP，蛋白质二硫键异构酶和其他重要的质量控制因素的作用，一个基于SILAC的蛋白质组学方法将被用来发现其他ER驻留蛋白质的新贡献。利用有关HexA降解机制的信息，我将专门改变ER质量控制，以增加突变HexA的溶酶体活性。对突变型HexA的周转途径的更深入理解将允许使用可以穿过血脑屏障并特异性改变在HexA产生中重要的质量控制因子而不引起全局ER应激或影响一般蛋白质折叠和ERAD的药物。