AUTO-UBIQUITYLATION · Structural insight into auto-ubiquitylation as a regulatory mechanism for protein trafficking
7РП — „Хора“ (Действия „Мария Кюри“)
- Период
- 2008-09-01 → 2011-08-31
- Финансиране от ЕС
- 75 000 €
- Участници
- 1
- Схема
- MC-IRG
Линиите свързват координатора с партньорите.
Накратко на български
Процесът на убиквитинилация регулира съдбата на протеините, като например при протеина Vps9 той променя структурата му. Разработването на метод за синтезиране на тези протеини в бактерии помага за точното определяне на тяхната структура и функция.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
Structural insight into auto-ubiquitylation as a regulatory mechanism for protein trafficking
We developed a synthetic biology approach that reconstitutes the entire ubiquitylation cascade in bacteria. Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and bio- physical characterisation of ubiquitylated proteins. We cir- cumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiqui- tylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterisation of several ubiquitylated proteins demon- strated the integrity, specificity and fidelity of the system, and revealed new biological findings. Recently, we also obtained crystal of ubiquitylated protein and were able to determined its structure. This is the first structure of ubiquitylated protein other than Ub itself, i.e. ubiquitylated-Ub.
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
The cell interprets the information conferred by ubiquitin (Ub) through Ub-receptors, proteins that harbor ubiquitin-binding domains (UBDs). Ub-receptors can be found in three mutually exclusive forms. Apo is the unbound form, whereas trans is the intermolecular form bound to ubiquitylated cargo. Intriguingly, the third form, cis, is of Ub-receptors that are themselves ubiquitylated. Monoubiquitylation of the Ub-receptors imposes an auto-inhibitory conformation, rendering them unable to bind ubiquitylated cargos, and thus providing an intrinsic regulatory mechanism. While several structures of apo and trans are available, a cis structure form of an ubiquitylated Ub-receptor has yet to be determined. Determining a cis structure is crucial for understanding the molecular mechanism of auto-inhibition. Due to the action of deubiquitylating enzymes, the ubiquitylated form of the Ub-receptors is labile and therefore it is difficult to purify them from eukaryotes. To fill this gap in knowledge, we developed a novel bacterial system for protein ubiquitylation. The system produces a high yield of ubiquitylated protein that can be easily purified and subjected to crystallization trials. Using this bacterial ubiquitylation system, we will determine cis as well as apo and trans structures of Ub-receptors. Guided by comparison of the structures, point mutations will be introduced into key residues. In vitro functional studies including the determination of oligomeric stages, and affinity measurements using isothermal titration calorimetry and analytical ultracentrifugation will be performed on all three forms. Finally, the phenotype of the wild type and mutant proteins will be studied in vivo by monitoring trafficking of GFP-cargo under confocal microscopy. The outcome of this study is predicted to provide the molecular principles of ub-receptor regulation, and serve to design drugs for diseases that involve the monoubiquitin pathways such as AIDS and cancer.
Оригинален текст от CORDIS (на английски).
Участници
- TEL AVIV UNIVERSITY · Tel AvivКоординаторИзраел
Връзки
Данни: CORDIS, © Европейски съюз