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.

Public Health Relevance

Tay-Sachs disease is a lysosomal storage disorder (LSD) in which mutations in the enzyme ?-hexosaminidase A (HexA) lead to toxic buildup of GM2 ganglioside and cell death, particularly in the neurons of patients. Like many LSDs, this disease is caused by recessive genetic mutations that give rise to unstable, but active, enzyme which is trapped by the endoplasmic reticulum (ER) quality control system before it can properly localize to the lysosome. This proposal aims to define the role of ER molecular chaperones and degradation factors necessary for the sensing and disposal of mutant HexA, with the ultimate goal of regulating quality control factors to specifically promote release of mutant HexA without globally affecting the state of the ER.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-F03B-A (20))
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Morris, Jill A
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University of Pennsylvania
Schools of Medicine
United States
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Dersh, Devin; Jones, Stephanie M; Eletto, Davide et al. (2014) OS-9 facilitates turnover of nonnative GRP94 marked by hyperglycosylation. Mol Biol Cell 25:2220-34