Apolipoprotein B (ApoB) is the major structural protein component of chylomicrons and very low and low density lipoproteins, the major cholesterol carrying particles in the blood. High levels of ApoB have been directly linked to the risk of developing coronary artery disease. Pre-secretory degradation is one important mechanism regulating the assembly and secretion of ApoB.
A major pre-secretory degradation pathway regulating ApoB production is Endoplasmic Reticulum Associated Degradation (ERAD), a pathway in which molecular chaperones play key roles. Molecular chaperones assist with protein folding, assembly, translocation, and targeting misfolded proteins to the proteasome for degradation. Previous work in mammalian cells and using cell-free systems with yeast cytosols established that the cytosolic chaperones, Hsp70 and Hsp90, promote ApoB ERAD.
To identify additional chaperones involved in ApoB ERAD, I employed a cell-free system in which Hsp70, Hsp90 and proteasome-dependent degradation of the ApoB48 isoform is recapitulated with yeast cytosol. In addition, I developed an in vivo yeast expression system for the ApoB29 isoform. The shorter ApoB29 isoform is localized to the ER and associates with chaperones in yeast. Using both the in vitro and in vivo systems I found that a yeast Hsp110, Sse1p, associates with and stabilizes ApoB, which contrasts with data indicating that Hsp70 and Hsp90 facilitate ApoB degradation. Because Sse1p is reported to associate with Ssa1p, an Hsp70 known to regulate ApoB degradation in vitro, and Ssb1p and Ssb2p, Hsp70s in the ribosome associated complex (RAC), I tested the contributions of Ssb1p and Ssb2p on ApoB ERAD in vitro and in vivo but observed no difference in ApoB degradation rates compared to wildtype strains. Together, these data indicate that Sse1p, but not RAC, contributes to ApoB stabilization. To determine whether my results are relevant in mammalian cells, Hsp110 was over-expressed in hepatocytes and enhanced ApoB secretion was observed. This study indicates that chaperones within distinct complexes can play unique roles during ER-associated degradation (ERAD), establishes a role for Sse1/Hsp110 in ERAD, and identifies Hsp110 as a target to lower cholesterol.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-03202008-152922 |
| Date | 12 June 2008 |
| Creators | Hrizo, Stacy Lynn |
| Contributors | Ora Weisz, Karen Arndt, William Saunders, Jeffrey Hildebrand, Jeffrey Brodsky |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-03202008-152922/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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