التفاصيل البيبلوغرافية
| العنوان: |
The roles of proteasome-associated factors RPN13, UCHL5, and UBE3C in basal, unfolded, and aggregated protein turnover |
| المؤلفون: |
Panov, Alexandra |
| المساهمون: |
Harper, J. Wade, Finley, Daniel, Denic, Vladimir, Feany, Mel, Raman, Malavika |
| سنة النشر: |
2024 |
| المجموعة: |
Harvard University: DASH - Digital Access to Scholarship at Harvard |
| مصطلحات موضوعية: |
hul5, proteasome, RPN13, UBE3C, Uch37, UCHL5, Biology, Molecular biology |
| الوصف: |
Posttranslational modifications, such as ubiquitin, allow cells to rapidly adapt to various cellular states by regulating protein stability, localization, and activity. A previous collaborative study between my lab and the Kopito Lab found that UBE3C, a HECT E3 ligase, is immediately ( 10 min) recruited to the proteasome upon chemically induced misfolding of a single aggregation-prone protein, agDD, suggesting a potential quality control mechanism during acute protein unfolding. Once recruited to the proteasome, UBE3C monoubiquitylates proteasomal ubiquitin receptor RPN13 (also known as ADRM1). However, the functional consequence of these events during protein aggregation clearance is unknown. To determine the functional consequences of UBE3C recruitment and RPN13 ubiquitylation, I developed new tools and approaches to study protein unfolding and aggregate turnover using the constructs DD and agDD, respectively. Both DD and agDD are maintained as a soluble, diffuse pool when cells are cultured in the presence of a small molecule ligand, Shield-1 (S1). Upon S1 washout, while DD immediately unfolds, agDD immediately unfolds and begins to aggregate, and both constructs simultaneously compete with cellular ubiquitylation and degradation quality control machinery. To directly assess the role of UBE3C, RPN13, and RPN13 ubiquitylation during agDD unfolding and aggregation, I used CRISPR/Cas9 gene editing to generate RPN13 and UBE3C knockouts and RPN13KR homozygous ubiquitylation site mutants in mammalian cell lines. I characterized these perturbations by combining agDD and DD expression with quantitative flow cytometry and biochemical approaches. In addition, I investigated the role of RPN13 and UBE3C in basal protein turnover using quantitative mass spectrometry-based proteomics and regression modeling. In Chapter 2, I show first that agDD turnover primarily occurs through the UPS and not autophagy or asymmetric cell division. I show that rapid turnover of agDD remains largely unaffected in RPN13KR cells, indicating ... |
| نوع الوثيقة: |
thesis |
| وصف الملف: |
application/pdf |
| اللغة: |
English |
| تدمد: |
31296270 |
| Relation: |
Panov, Alexandra. 2024. The roles of proteasome-associated factors RPN13, UCHL5, and UBE3C in basal, unfolded, and aggregated protein turnover. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.; https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37378991; orcid:0000-0002-2960-0291 |
| الاتاحة: |
https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37378991 |
| رقم الانضمام: |
edsbas.84377105 |
| قاعدة البيانات: |
BASE |