PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer's amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE).
BACKGROUND
Alzheimer's disease (AD) is intimately tied to amyloid-β (Aβ) peptide. Extraneuronal brain plaques consisting primarily of Aβ aggregates are a hallmark of AD. Intraneuronal Aβ subunits are strongly implicated in disease progression. Protein sequence mutations of the Aβ precursor protein (APP) account for a small proportion of AD cases, suggesting that regulation of the associated gene (APP) may play a more important role in AD etiology. The APP promoter possesses a novel 30 nucleotide sequence, or "proximal regulatory element" (PRE), at -76/-47, from the +1 transcription start site that confers cell type specificity. This PRE contains sequences that make it vulnerable to epigenetic modification and may present a viable target for drug studies. We examined PRE-nuclear protein interaction by gel electrophoretic mobility shift assay (EMSA) and PRE mutant EMSA. This was followed by functional studies of PRE mutant/reporter gene fusion clones.


RESULTS
EMSA probed with the PRE showed DNA-protein interaction in multiple nuclear extracts and in human brain tissue nuclear extract in a tissue-type specific manner. We identified transcription factors that are likely to bind the PRE, using competition gel shift and gel supershift: Activator protein 2 (AP2), nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF), and specificity protein 1 (SP1). These sites crossed a known single nucleotide polymorphism (SNP). EMSA with PRE mutants and promoter/reporter clone transfection analysis further implicated PuF in cells and extracts. Functional assays of mutant/reporter clone transfections were evaluated by ELISA of reporter protein levels. EMSA and ELISA results correlated by meta-analysis.


CONCLUSIONS
We propose that PuF may regulate the APP gene promoter and that AD risk may be increased by interference with PuF regulation at the PRE. PuF is targeted by calcium/calmodulin-dependent protein kinase II inhibitor 1, which also interacts with the integrins. These proteins are connected to vital cellular and neurological functions. In addition, the transcription factor PuF is a known inhibitor of metastasis and regulates cell growth during development. Given that APP is a known cell adhesion protein and ferroxidase, this suggests biochemical links among cell signaling, the cell cycle, iron metabolism in cancer, and AD in the context of overall aging.
Laboratory of Molecular Neurogenetics, Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, 791 Union Drive, Indianapolis, IN 46202, USA. dlahiri@iupui.edu
Debomoy K
DK
Lahiri
Bryan
B
Maloney
Jack T
JT
Rogers
Yuan-Wen
YW
Ge
D000544:Alzheimer Disease / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D015894:Genome, Human / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D054778:NM23 Nucleoside Diphosphate Kinases / Q000378:metabolism*; D009928:Organ Specificity; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics
D016564:Amyloid beta-Protein Precursor; D054778:NM23 Nucleoside Diphosphate Kinases; D004247:DNA; C517940:NME2 protein, human