Daskivich, Grant Joseph
(2024)
Characterization and contextualization of problematic substrates for Endoplasmic Reticulum Associated Degradation.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Proteostasis, the finely tuned system that ensures proper protein folding, trafficking, and clearance, is a fundamental process in all living organisms. This dynamic network of cellular pathways regulates the quality and quantity of proteins within the cell. It is crucial for maintaining cellular homeostasis and preventing the accumulation of aberrant proteins, which can lead to cellular dysfunction and disease. Specifically, highly stable protein aggregates irreversibly disrupt proteostasis and trigger disease onset. Therefore, for my research, I constructed TM-Ubc9ts, a temperature-sensitive membrane protein degraded by ER-associated degradation (ERAD) in yeast, to study how endoplasmic reticulum tethered, aggregation-prone proteins impact proteostasis. I hypothesized that TM-Ubc9ts would be an ERAD substrate in mammalian cells and aggregate at elevated temperatures.
In this document, I first review the proteostasis network, beginning with protein folding, followed by protein aggregation and finally protein quality control. I then explain how I tested the above hypothesis via cycloheximide chase and detergent solubility assays respectively. Degradation of TM-Ubc9ts was largely dependent on the proteasome, especially at elevated temperatures. Solubility did not change significantly, but I observed the evolution of distinct clipped forms of TM-Ubc9ts that were stabilized when the proteasome was inhibited. Subsequently, I ask if TM-Ubc9ts was a substrate of the intramembrane protease RHBDL4, which clips ER membrane proteins in preparation for retrotranslocation. Though I found TM-Ubc9ts clipping to be independent of RHBDL4 function, I established a pipeline by which ER membrane proteins can be characterized at specific stages of ERAD. Finally, I describe the rationale and construction of another set of aggregation-prone ERAD substrates, Sec62-Aβ42 and Sec62-Aβ40. These substrates were analyzed through the same experimental pipeline and found to be remarkably stable compared to TM-Ubc9ts, highlighting the complex relationship between hypotheses and experimental results. Future work can utilize this streamlined set of experiments to quickly and precisely identify the point at which aggregation prone proteins in the ER disrupt proteostasis.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
10 January 2024 |
| Date Type: |
Publication |
| Defense Date: |
19 September 2023 |
| Approval Date: |
10 January 2024 |
| Submission Date: |
6 September 2023 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
124 |
| 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: |
Protein homeostasis, Protein degradation, Aggregation |
| Date Deposited: |
10 Jan 2024 14:32 |
| Last Modified: |
10 Jan 2024 14:32 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/45384 |
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