A Study Of PEN-2 And Interacting Protein By Yeast Two-Hybrid

Alzheimer's disease (AD) is a degenerative disease that causes gradual loss of memory, judgment, and ability to function socially.Γ-secretase catalyzes the intramembrane proteolysis of Notch, beta-amyloid precursor protein (APP), and other substrates as part of a new signaling paradigm and as a key step in the pathogenesis of Alzheimer's disease. Theγ-secretase is a membrane protein complex comprised of Presenilin (PS), nicastrin (NCT), anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2). PEN-2 has been demonstrated to be crucial for the endoproteolysis of full-length, PS to generate the N-fragments and C-fragments, which is refolded for the stability and the achieving of the PS/γ-secretase. Recently, it has also been demonstrated that PEN-2 is involved in promoting neuronal cell survival and protecting neurons from apoptosis in vivo. However, much of pathophysiological functions of PEN-2 and the underlying mechanisms still remain elusive.To further understand the function of PEN-2 in the pathology of AD, we utilized PEN-2 as the bait to screen human fetal brain cDNA library using the yeast two-hybrid system.Twenty-five yeast positive clones were obtained. After sequencing, the positive clones were analyzed with BLAST. Four candidate proteins were chosen for further characterization: they are PCBP4 [Homo sapiens poly (rC) binding protein 4], TXNDC9 (Homo sapiens thioredoxin domain containing 9), FTL (Homo sapiens ferritin, light polypeptide) and GRK5 (Homo sapiens G protein-coupled receptor kinase 5). Theβ-galactosidase activity assay demonstrated that four proteins can interact with PEN-2 in yeast strain AH109. The recombinant plasmids of pCMV5-HA-PEN2,pCMV-myc-TXNDC9 and pCMV-myc-FTL were constructed successfully. For the expression of these proteins in mammalian cells, the expression of TXNDC9 and FTL in 293T cells has been confirmed. Our studies demonstrated that four proteins are able to interact with PEN-2 in the yeast strain.Our research may contribute to further understanding the function of PEN-2 which...