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Using VHHs to study the function and control of the E2 Ubiquitin-conjugating enzyme UBC6e
Başlık:
Using VHHs to study the function and control of the E2 Ubiquitin-conjugating enzyme UBC6e
Yazar:
Ling, Jingjing, author.
Yazar Ek Girişi:
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Hidde Ploegh.
Özet:
Endoplasmic reticulum-associated degradation (ERAD) is essential for protein quality control both during stress and at steady state. We found that an E2 ubiquitinconjugating enzyme UBC6e contributes to a new layer of homeostatic control, in which ERAD activity itself is regulated post-transcriptionally and independently of the unfolded protein response. Ablation of UBC6e causes up-regulation of active ERAD enhancers and increases clearance not only of terminally misfolded substrates, but also of wild-type glycoproteins that fold comparatively slowly. Tuning of ERAD component level involves a mechanism that is likely distinct from the conventional ERAD by Hrdl/SEL1L complex.
To better understand how UBC6e is controlled, we developed VHH05, which associates with UBC6e with a low nanomolar dissociation constant. VHHO5 enhances enzymatic activity of UBC6e by binding to a gateway helix on UBC6e (168-186). As the phosphorylation site S184 is on this gateway loop, we propose that stress-induced phosphorylation of UBC6e allows binding of a cytosolic factor that enhances UBC6e activity similarly to VHH05.
We further characterized the binding of VHHO5 to UBC6e to identify a 14mer binding epitope QADQEAKELARQIS. This epitope was translated into a 6e-tag recognized by VHH05. VHHO5 specifically retrieves 6e-tagged proteins from a complex lysate mixture. VHHO5 sortagged with biotin or fluorophores can stain 6e-tagged protein in an immunoblot and in FACS. VHHO5 recognizes 6e-tag in the reducing environment of the cytosol and can be used to target tagged proteins to selective locations in the cell.
To study the ubiquitination machinery in live cells, we used CellSqueeze technology to deliver epitope-tagged ubiquitin (Ub) to HeLa cells. The delivered Ub molecules are readily utilized in the cell. Furthermore, kinetics of Ub incorporation is much faster in lysates than in squeezed cells. Further experiments using E2s pre-loaded with Ub in the presence of El inhibition showed that E2-Ubs are also utilized faster in lysates than in squeezed cells. We hope to use this technology to identify the contribution of E2s to substrate selectivity and characterize interactions between E3s and E2-Ubs. (Copies available exclusively from MIT Libraries, libraries.mit.edu/docs - docs mit.edu).
Notlar:
School code: 0753
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(687488.1) | 687488-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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