Abstract
BackgroundAlzheimer’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.ResultsEMSA 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.ConclusionsWe 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.
Highlights
Alzheimer’s disease (AD) is intimately tied to amyloid-β (Aβ) peptide
Restricting predicted sites to mammal TF matrices and eliminating redundant sites resulted in predicted affinities with several transcription factors (Table 1), including Activator protein 2 (AP2), GATA binding proteins 1 (GATA1) and 2 (GATA2), two GC boxes, paired box gene 4-a (Pax4a), nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF), EpsteinBarr virus transcription factor R (R), specificity protein 1 (SP1), and transcription elongation regulator 1
DNA-protein interaction of the proximal regulatory element” (PRE) by electrophoretic mobility shift assay (EMSA) varies among tissue types and cell line conditions To investigate cell type specificity of PRE-protein interactions, we used the PRE fragment in EMSA with nuclear extracts from rat pheochromocytoma cells and extracts (PC12) and human cervical epithelial (HeLa), SK-N-BE neuroblastoma (NB), and histiocytic lymphoma (U937) cells (Figure 1A–C) and with nuclear extracts from human tissues (Figure 1D)
Summary
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. 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. A diagnostic feature of Alzheimer’s disease (AD) is aggregation of toxic amyloid β peptide (Aβ) into extracellular plaques, suspected of causing or contributing to disease progression [1,2]. The more upstream of these two polymorphic sites may function as a target site for Aβ acting as a transcription factor [4,5]
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