A Study Of Nicastrin And Interacting Protein By Yeast Two-Hybrid

γ-secretase is a high molecular weight complex comprises of at least four components, presenilin (PS), nicastrin (NCT), anterior pharynx defective-1 (APH-1) and presenilin enhancer-2 (PEN-2). The cleavage ofβ-amyloid precursor protein (APP) byγ-secretase releases small peptides calledβ-amyloid (Aβ), which is the prime culprit for the pathogenesis of Alzheimer's disease (AD). In addition,γ-secretase catalyzes the intramembrane proteolysis of many other functionally important proteins such as Notch, Cadherin, ErbB4, etc. Among theseγ-secretase components, NCT has been found to be crucial for the binding of substrates. In addition, NCT is important for the stability and proper intracellular trafficking of otherγ-secretase components. To further understand the patho/physiological functions of NCT, we utilized a yeast-two-hybrid assay to screen human fetal brain cDNA library and identify proteins that can interact with NCT.Initial screening obtained 19 positive yeast clones. After sequencing and BLAST search, we identified two candidate proteins as MT2A (metallothionein-2A) and THAP7 (THAP domain-containing protein 7). Theβ-galactosidase activity assay demonstrated that both proteins can interact with NCT in yeast strain AH109. Further study in the mammalian 293T cells showed that a co-immunoprecipitation of MT2A and NCT.MT2A belongs to the metallothionein (MT) family proteins. The levels of two MT family members, MT-I and MT-II, are dramatically up-regulated by neuroinflammation. In AD patients, immunostaining of MT-I and MT-II is also dramatically increased in cells surrounding the amyloid plaques. These results including ours suggest that MT family proteins may play important roles in the pathogenesis of AD, possibly through the interaction with NCT to alter the activity/cleavage ofγ-secretase. Further study along this way may elucidate the underlying mechanisms.