NCT-BINDING · Substrate docking and affinity studies of Nicastrin: the binding mechanism
6РП — Действия „Мария Кюри“
- Период
- 2007-10-01 → 2009-09-30
- Финансиране от ЕС
- 153 319 €
- Участници
- 1
- Схема
- IIF
Линиите свързват координатора с партньорите. За проекти отпреди 2014 г. CORDIS не винаги дава точни координати. Тези точки са на ниво град или държава.
Накратко на български
Никастринът и неговата роля при разграждането на протеини, като например този, свързан с болестта Алцхаймер, се анализират чрез промяна на определени аминокиселини. Това помага да се разбере как се сглобява комплексът гама-секретаза, вместо как той разпознава своите субстрати.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
Final Activity Report Summary - NCT-BINDING (Substrate docking and affinity studies of Nicastrin: the binding mechanism.)
The gamma-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin (NCT) was proposed to operate as the substrate receptor of the complex with the glutamate 332, Glu(333) in humans, serving as the anionic binding site for the alpha-amino-terminal group of substrates. The putative binding site was located within the aminopeptidase-like domain of NCT. The Glu(332) was proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu(332) could bind the alpha-amino-terminal group of substrates, we hypothesised, in analogy with M28-aminopeptidases, that other residues in the putative binding site of NCT should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of amyloid precursor protein (APP) and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolished the anionic alpha-amino-terminal binding function, remained fully active. When we introduced the previously characterised E332A mutation, we found strongly decreased gamma-secretase complex levels; nevertheless the remaining complex appeared as active as the wild-type complex. We confirmed, in two independent in vitro assays, that the specific enzymatic activity of E332A mutant was comparable to that of the wild-type complex. Thus, Glu(332) crucially affected complex maturation rather than substrate recognition. Moreover, other NCT mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally gamma-secretase activity. Consequently, these studies indicated that the main role of Glu(332) relied in the maturation and assembly of gamma-secretase rather than in the substrates recognition. Even though our experiments did not rule out the fact that the ectodomain of NCT also participated in the enzymatic function of the gamma-secretase complex, our results clearly demonstrated that the previously proposed mechanism could not be maintained as an explanation for the available data. Hence, a discussion was reopened regarding NCT function in the gamma-secretase-mediated regulated intramembrane proteolysis.
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
Beta-Secretase (beta-SEC) is responsible for the intramembrane cleavage of amyloid precursor protein, an event intimately linked to Alzheimer's disease (AD). Beta-SEC is an aspartic protease composed by presenilin, nicastrin (NCT), APH-1, and PEN-2.Beta-SEC appears to have a very broad specificity; however, no quantitative data is available to support this assumption. NCT subunit is a type 1 membrane protein whose ectodomain (NCT ECD) actively participates in the substrate binding. NCT-substrate interaction is an essential step in the beta-SEC mechanism.The characterization of this interaction is important to better understand the secretase specificity, but also because it represents an attractive point for the design of inhibitors.The project objectives are: determine structure-functional relationships (SFRs) to reveal the key functionalities in the molecular structure of the substrates that are responsible for recognition by NCT and therefore for beta-SEC activity or inhibition; evaluate the contribution of NCT on the complex specificity; generate and evaluate a entirely new class of beta-SEC inhibitors.The NCT ECD is reminiscent of aminopeptidase structures and the NCT-E333 function suggests a common binding mechanism. Taking advantage of this similarity, we will build a model for the NCT ECD to define and explore a putative binding pocket.BIAcore technology and isotermal titration microcalorimetry will be used to characterize the interaction between NCT ECD (wt or mutant) and the substrate ectodomains.To gain further insight into which functional groups are significant for substrate recognition we will use a set of compounds to investigate the effects on constants affinities, with special interest in inhibitory activities.In conclusion, we will generate quantitative data about the specificity (related to NCT) of the beta-SEC. The experimentally determined SFRs will shed light on the possibility to design novel and more selective inhibitors for the AD's amyloid cascade.
Оригинален текст от CORDIS (на английски).
Участници
- FLANDERS INTERUNIVERSITARY INSTITUTE FOR BIOTECHNOLOGY VZW · ZWIJNAARDEКоординаторНиво градБелгия
Връзки
Данни: CORDIS, © Европейски съюз