A common theme that is emerging from studies of neurodegenerative disorders is the involvement of misfolded proteins and defects in the ubiquitin-proteasome system. The ubiquitin-proteasome system was originally considered to be the machinery for tagging and destroying unwanted proteins. However, recent evidence indicates that the ubiquitin-proteasome system is also involved in protein unfolding, intracellular protein targeting, cell signaling and transcription. Our laboratory identified ubiquilin, the founding member of an exciting new class of proteins, which appears to inhibit degradation of proteins. Ubiquilin contains multiple ubiquitin-related motifs typically found in proteins involved in the ubiquitin-proteasome system. We identified ubiquilin through its interactions with presenilin proteins, mutations in which are associated with early-onset familial Alzheimer's disease. Overexpression of ubiquilin in cells increases presenilin protein levels, decreases levels of endoproteolytic N- and C-terminal presenilin fragments, and decreases ubiquitination of presenilin proteins. Several lines of evidence, including results from our laboratory, suggest that ubiquilin expression is induced during cell stress and that it may function as a molecular chaperone, a ubiquitin-receptor, or in cell survival. We propose to use a multi-pronged approach to determine the role ubiquilin proteins play in cells and organisms. Using cellular, molecular, and immunological techniques, we will determine the expression patterns of the different ubiquilin proteins in tissues and tissue culture cells as well as the intracellular localization properties of different ubiquilin isotypes. We will determine how different domains of the ubiquilin polypeptide are involved in the functions of the protein. We will use both transfection assays, as well as an in vitro cell-free translation assay, to identify the role ubiquilin plays in the ubiquitin-proteasome system. We will characterize ubiquilin-interacting proteins using co-immunoprecipitation and yeast two-hybrid assays. Finally, we propose to use gene knockout in mouse, anti-sense inhibition in C. elegans and human tissue culture cells, and antibody neutralization to identify the effects that loss of ubiquilin has in cells and organisms. The results obtained from the proposed research will lead to a better understanding of the functional role of ubiquilin in cells and in whole organisms, and ultimately, its role in health and in disease.
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