Doonan, Lynley
(2018)
UNDERSTANDING APOLIPOPROTEIN B’S ABILITY TO AGGREGATE THROUGH LIPID DROPLETS AND CHAPERONE HOLDASE.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Endoplasmic Reticulum (ER) associated degradation (ERAD) is the general process in which misfolded secretory proteins are monitored and degraded to protect the cell from a buildup of nonfunctioning proteins. Apolipoprotein B (ApoB), an ERAD substrate is a large hydrophobic secretory protein associated with the transport of lipids and cholesterol by lipoproteins in the body. ApoB synthesis involves cotranslational translocation through the Sec61 translocon into the ER. If properly folded and lipidated, ApoB is then retrotranslocated through the same pore. Since ApoB contains many aggregation-prone hydrophobic β-sheets, what prevents ApoB aggregation before degradation by ERAD? Initial considerations suggested that cytosolic factors, such as lipid droplets or chaperone “holdases,” “foldases,” and “disaggregases” may help to maintain ApoB’s solubility post retrotranslocation. To test this hypothesis, I adapted our yeast galactose inducible ApoB expression system to be β-estradiol inducible and used it to investigate various chaperone candidates to determine if they affect ApoB stability. Upon large scale isolation of lipid droplets, ApoB was found not to interact with lipid droplets. Next, I investigated potential chaperones. I found that the small heat shock proteins, a family of ATP-independent chaperones, and the TRiC complex, an Hsp60 family member, do not affect ApoB stability. However, I determined that Hsp104, a AAA+ ATPase which helps to refold and reactivate aggregated proteins, is a pro-degradation factor for ApoB. ApoB degradation was slowed in the absence of this chaperone while overexpression caused faster degradation. I then investigated Rvb2, the yeast homolog of the human functional analog of Hsp104, to determine its effect on ApoB stability. Unexpectedly, Rvb2 did not restore ApoB degradation in the absence of Hsp104. Together, my data indicate that ApoB does require chaperone disaggregase function prior to ERAD.
Share
| Citation/Export: |
|
| Social Networking: |
|
Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
31 January 2018 |
| Date Type: |
Publication |
| Defense Date: |
1 August 2017 |
| Approval Date: |
31 January 2018 |
| Submission Date: |
7 December 2017 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
181 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Cardiovascular Disease, Cholesterol, ApolipoproteinB, Very Low Density Lipoprotein,
Endoplasmic Reticulum Associated Degradation, Autophagy |
| Date Deposited: |
31 Jan 2018 15:57 |
| Last Modified: |
31 Jan 2018 15:57 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/33590 |
Metrics
Monthly Views for the past 3 years
Plum Analytics
Actions (login required)
 |
View Item |
![[feed]](/images/feed-icon-32x32.png){kind=icon}