| Autor: |
Keren‐Kaplan, Tal, Attali, Ilan, Estrin, Michael, Kuo, Lillian S, Farkash, Efrat, Jerabek‐Willemsen, Moran, Blutraich, Noa, Artzi, Shay, Peri, Aviyah, Freed, Eric O, Wolfson, Haim J, Prag, Gali |
| Předmět: |
|
| Zdroj: |
EMBO Journal; 2/13/2013, Vol. 32 Issue 4, p538-551, 14p, 4 Diagrams, 1 Chart, 4 Graphs |
| Abstrakt: |
The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V:Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a Kd of 119 μM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC50 of 27.6 μM and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
|
Plný text