Kumari, Deepa
(2021)
Investigating the roles of Hsp40s and an E3 ubiquitin ligase in the biogenesis of Apolipoprotein B.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Apolipoprotein B (ApoB) is the primary component of atherogenic lipoproteins and is responsible for the transport of dietary and endogenous fats and cholesterol in the blood stream. Elevated levels of ApoB-containing particles lead to the development of Coronary Artery Disease, which kills millions of people every year. As it is synthesized, ApoB is co-translationally translocated into the endoplasmic reticulum (ER) through the Sec61 translocon and is lipidated, leading to the formation of a lipoprotein particle that is eventually secreted into the plasma. However, in the absence of lipidation, ApoB is retrotranslocated to the cytosol and destroyed via ER Associated Degradation (ERAD), a regulated pathway that maintains proteostasis by normally degrading misfolded proteins in the early secretory pathway. In this thesis, I investigated the contributions of select cytosolic Hsp40s on ApoB biogenesis. To this end, I used a rodent cell line that endogenously synthesizes and secretes ApoB. By performing siRNA mediated knockdown and metabolic pulse chase experiments, I discovered that a class A Hsp40, DNAJA1, associates with and facilitates ApoB degradation. Consistent with these data, Ydj1 (the yeast homolog of DNAJA1) also facilitates ApoB degradation in a heterologous yeast expression system. The ablation of DNAJA1 also leads to the accumulation of ubiquitinated ApoB, suggesting that DNAJA1 functions prior to degradation. In stark contrast to DNAJA1, DNAJB1, a class B member, stabilizes ApoB. Because knockdown decreases the levels of ubiquitinated ApoB, DNAJB1 might act prior to DNAJA1 as ApoB enters the ER. Finally, because ERAD substrates must be ubiquitinated after chaperone-dependent selection, I examined the contributions of E3 ubiquitin ligases on ApoB stability and discovered that Hrd1 targets ApoB for degradation in the rodent cell line. Together, my findings highlight the unexpected functional diversity of Hsp40s during the biogenesis of ApoB.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
8 October 2021 |
| Date Type: |
Publication |
| Defense Date: |
7 June 2021 |
| Approval Date: |
8 October 2021 |
| Submission Date: |
15 July 2021 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
187 |
| 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: |
Apolipoprotein B, Coronary Artery Disease, Hsp40, E3 ubiquitin ligase |
| Date Deposited: |
08 Oct 2021 19:51 |
| Last Modified: |
08 Oct 2022 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/41335 |
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