PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer's amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE)

Debomoy Lahiri, Bryan Maloney, Jack T. Rogers, Yuan Wen Ge

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article number68
JournalBMC Genomics
Volume14
Issue number1
DOIs
StatePublished - Jan 31 2013

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Amyloid beta-Protein Precursor
Protein Precursors
Electrophoretic Mobility Shift Assay
Alzheimer Disease
Proteins
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Clone Cells
Gels
Transfection
NM23 Nucleoside Diphosphate Kinases
Transcription Factors
Enzyme-Linked Immunosorbent Assay
Calcium-Calmodulin-Dependent Protein Kinases
Ceruloplasmin
Tissue Extracts
Transcription Initiation Site
Gene Fusion
Brain
Protein Kinase Inhibitors
Nuclear Proteins

Keywords

  • Alzheimer's disease
  • Amyloid precursor protein
  • Cancer
  • Gene regulation
  • Gene transcription
  • Iron
  • Latency
  • nm23 nucleoside diphosphate kinase
  • Oncogenesis
  • PuF
  • SP1
  • Specificity protein 1
  • Transcription factor

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer's amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE). / Lahiri, Debomoy; Maloney, Bryan; Rogers, Jack T.; Ge, Yuan Wen.

In: BMC Genomics, Vol. 14, No. 1, 68, 31.01.2013.

Research output: Contribution to journalArticle

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T1 - PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer's amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE)

AU - Lahiri, Debomoy

AU - Maloney, Bryan

AU - Rogers, Jack T.

AU - Ge, Yuan Wen

PY - 2013/1/31

Y1 - 2013/1/31

N2 - 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.

AB - 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.

KW - Alzheimer's disease

KW - Amyloid precursor protein

KW - Cancer

KW - Gene regulation

KW - Gene transcription

KW - Iron

KW - Latency

KW - nm23 nucleoside diphosphate kinase

KW - Oncogenesis

KW - PuF

KW - SP1

KW - Specificity protein 1

KW - Transcription factor

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