Functional analysis of proteins, which interact with Alzheimer's disease amyloid precursor protein and presenilins

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1.Using the yeast two-hybrid system, we screened for proteins interacting with APP-binding protein Fe65L2 and cloned the cDNA large subunit of RNA polymerase 2 (RPB1). The cDNA encoded the RPB1 C-terminal domain containing the repeat domain consisting of 52 repeats of seven amino acids. Fe65L2 preferentially interacted with hypo-phosphorylated form of RPB1. Reporter gene assay using GAL4-dependent reporter gene and Fe65L2-GAL4 DNA-binding domain fusion showed transcriptional suppression. These results suggest that Fe65L2 forms complexes with RNA polymerase 2 in nuclei and regulates transcription.2.Gel shift assay using DNA probe containing c954C>T polymorphism sequence indicated the existence of nuclear protein interacting with the probe sequence.3.Overexpression of PS2-binding protein in HEK293 cells that stably overe pressed APP, DRAL increased the secretion of soluble APP and decreased the secretion of Aβ, suggesting that DRAL affect the metabolism of APP.4.We found a novel splicing variant of BACE, 1-127 which is regulated by nonsense-mediated mRNA decay (NMD). This transcript was slightly detected in the human brain. When expressed in cells, 1-127 is retained in ER without its propeptide removal, unstable and degraded by proteasome-dependent manner. Thus 1-127 is regulated by both NMD and proteasome-dependent degradation.