A genetic approach to the role of the ubiquitin /proteasome pathway in neurodegeneration.
Item
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Title
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A genetic approach to the role of the ubiquitin /proteasome pathway in neurodegeneration.
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Identifier
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AAI3083682
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identifier
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3083682
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Creator
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Li, Zongmin.
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Contributor
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Adviser: Maria E. Figueiredo-Pereira
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Date
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2003
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Cell
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Abstract
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Involvement of the ubiquitin/proteasome pathway in neurodegeneration has gained support from a variety of studies. Although selective sets of neurons are affected in different neurodegenerative disorders, they share a common pathological feature: the occurrence of intracellular inclusions containing ubiquitinated proteins. Under homeostatic conditions, ubiquitinated proteins do not accumulate in cells since they undergo selective proteolysis by the ubiquitin/proteasome pathway. The inability to eliminate ubiquitinated proteins may result from a malfunction or overload of the ubiquitin/proteasome pathway or from structural changes in the protein substrates halting their degradation.;Our studies investigate the possibility that proteasome dysfunction is an important contributor to neurodegeneration. We previously established a mouse neuronal cell model in which proteasome inhibitors induced the formation of intracellular inclusions containing ubiquitinated proteins and lead to cell death. In the present study, disruption of proteasome activity was achieved by mutating the active site of its MC13 catalytic subunit. MC13, the beta5 subunit of the mouse 20S proteasome, is responsible for the chymotrypsin-like activity that carries out the rate-limiting step in protein degradation through this pathway. The N-terminal Thr1 of MC13 is essential for its activity and was mutated to Alal. MC13 and its mutant were tagged with a c-myc epitope. The fusion proteins were subsequently expressed in mouse neuronal HT4 cells and stable transfectants were selected with hygromycin B. Under homeostatic conditions, expression of the MC13 mutant (T1A) in HT4 cells impaired proteasome chymotrypsin-like activity without being dominant lethal. However, expression of the MC13 mutant hypersensitized the cells to oxidative stress resulting in the accumulation of ubiquitinated proteins in aggregates and leading to cell death. These findings clearly establish that selective proteasome dysfunction causes neuronal changes that are common to a broad range of human neurodegenerative disorders. The genetic manipulation of proteasome activity in the neuronal cells provided the basis for establishing a transgenic mouse model to study the long-term effect of moderate proteasome dysfunction. Future studies with the cellular and the transgenic mouse models will provide important insights into the role of proteasome dysfunction in neurodegeneration.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
