Date of Award

7-13-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Jane R. Dunlevy

Abstract

BOG25 contains multiple protein-protein interaction domains at the N-terminus that are characteristic of the EH (Eps15 Homology) network family of proteins involved in endocytosis and a C-terminal death domain characteristic of proteins involved in apoptosis. This combination of protein-protein interaction domains had not been identified in other known proteins until a National Center for Biotechnology Information (NCBI) human genome BLAST search using the BOG25 amino acid sequence identified a novel gene, 7a5. The current study hypothesized that: (1) BOG25 and 7a5 are related proteins; and (2) the BOG25 death domain is biologically active and has a functional role in apoptosis in human retinal pigment epithelial (ARPE-19) cells. The results of this study demonstrated that BOG25 and 7a5 are related proteins with similar gene structures, domain homologies, amino acid homologies and localization patterns. 7a5 displayed a 60% homology to BOG25 and homology increased to 75% at the C-terminal death domain. The full-length BOG25 fusion protein was shown to localize to the plasma membrane and peripheral nucleus, while the full-length 7a5 fusion protein localized primarily to the basolateral region and vesicle-like structures. Co-localization was visualized primarily at the basolateral region of ARPE-19 cells by confocal microscopy. Distinct changes in cellular and nuclear morphology were characterized between the full-length, N-terminal and C-terminal BOG25 fusion proteins by confocal and transmission electron microscopy (TEM). ARPE-19 cells expressing the C-terminal BOG25 fusion proteins displayed a higher frequency of cellular and nuclear morphological changes compared to the other BOG25 fusion proteins and control conditions. Cells expressing the C-terminal BOG25 fusion protein displayed a rounded, spherical cell morphology and an increased percentage of nuclear condensation following analysis by confocal microscopy. TEM confirmed the nuclear condensation and identified an increase in autophagy. Treatment of ARPE-19 cells with the autophagy inhibitor, 3-methyladenine, reversed the dramatic cellular and nuclear morphological changes displayed by cells expressing the C-terminal BOG25 fusion protein. In conclusion, the results of this study demonstrated that BOG25 and 7a5 are related proteins, characterized the BOG25 C-terminal death domain in ARPE-19 cells and showed that the distinct cellular and nuclear morphological changes were characteristic of autophagy.

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