The Alzheimer's amyloid β-peptide (Aβ) binds a specific DNA Aβ-interacting domain (AβID) in the APP, BACE1, and APOE promoters in a sequence-specific manner: characterizing a new regulatory motif - PubMed (original) (raw)

The Alzheimer's amyloid β-peptide (Aβ) binds a specific DNA Aβ-interacting domain (AβID) in the APP, BACE1, and APOE promoters in a sequence-specific manner: characterizing a new regulatory motif

Bryan Maloney et al. Gene. 2011.

Abstract

Deposition of extracellular plaques, primarily consisting of amyloid β peptide (Aβ), in the brain is the confirmatory diagnostic of Alzheimer's disease (AD); however, the physiological and pathological role of Aβ is not fully understood. Herein, we demonstrate novel Aβ activity as a putative transcription factor upon AD-associated genes. We used oligomers from 5'-flanking regions of the apolipoprotein E (APOE), Aβ-precursor protein (APP) and β-amyloid site cleaving enzyme-1 (BACE1) genes for electrophoretic mobility shift assay (EMSA) with different fragments of the Aβ peptide. Our results suggest that Aβ bound to an Aβ-interacting domain (AβID) with a consensus of "KGGRKTGGGG". This peptide-DNA interaction was sequence specific, and mutation of the first "G" of the decamer's terminal "GGGG" eliminated peptide-DNA interaction. Furthermore, the cytotoxic Aβ25-35 fragment had greatest DNA affinity. Such specificity of binding suggests that the AβID is worth of further investigation as a site wherein the Aβ peptide may act as a transcription factor.

Copyright © 2011 Elsevier B.V. All rights reserved.

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Figures

Figure 1

Figure 1. Presence of Aβ–binding motifs (AβID) with 80% homology to p53 HSE sequence on APOE, APP, and BACE1 5’–flanking sequences

The 5’–flanking sequences of the APOE, APP, and BACE1 genes were searched for decamers with at least 80% homology to the “GGATTGGGGT” Aβ–binding HSE site of the TP53 promoter (Ohyagi et al., 2005). Sites marked with “*” were chosen for further study in this report.

A.

A region of the APOE gene from 2kb upstream of the +1 TSS to the end of the first coding exon, which contains the first intron, (Paik et al., 1985; Du et al., 2005) was searched.

B.

The APP 5’–flanking region from 4kb upstream of the +1 TSS to the end of the first coding exon (Lahiri and Robakis, 1991; Hattori et al., 1997) was searched.

C.

The BACE1 5’ flanking region from 3.8kb upstream of the +1 TSS to the “ATG” start codon (Christensen et al., 2004; Sambamurti et al., 2004) was searched.

Figure 2

Figure 2. Electrophoretic mobility shift assay (EMSA)/gel shift assay of Aβ1–42 vs. different putative binding decamer–containing oligomers

Figure shows representative EMSA of the

A.

Aβ1–42 and the

B,

Aβ1–40 peptides. The reverse Aβ40–1 and 42–1 peptides were independently incubated with radiolabeled oligomer containing the putative AβID at −3833 (lane 1). Aβ1–42 or 1–40 (lanes 2–16) were incubated with radiolabeled oligomers that contained the putative AβIDs in the APP (lanes 2–8) 5’–flanking region at positions −3833G (lane 2), −3833A with a “G” to “A” substitution at −3829 (lane 3), −3364 (lane 4), −2871 (lane 5) and −1862 (lane 6). In addition, an oligomer corresponding to the FAD mutation site in APP at −1023 (lane 7) and a distinct HSE–binding site within APP (lane 8) were incubated and run; in the APOE (lanes 9–12) 5’–flanking region at positions −899 (lane 9), +171 (lane 10), +284 (lane 11), +660/+665 (lane 12); and in the BACE1 (lanes 13–16) 5’–flanking region at positions −1939 (lane 13), −1766 (lane 14), −119 (lane 15), and +36 (lane 16). The gel was dried and exposed to X–ray film for autoradiography. DNA–peptide interactions appeared as dark signal near the top of the gel, unbound oligomer ran to the bottom of the gel.

Figure 3

Figure 3. Determination of “binding” vs. “non–binding oligomer pairs to Aβ peptides in EMSA

EMSA films were densitometrically scanned and signals within each film were normalized by subtracting the mean of that signal’s film from the individual signal and dividing by the standard deviation of signals. Normalized signals were then analyzed by Waller–Duncan means separation, and categories indicated with letters on the figure. Samples sharing the same letter were not significantly different at k = 100 (analogous to α = 0.05). The two “background control” oligomer pairs, _APP_HSE and _APP_−1023, indicated by “*”.

Figure 4

Figure 4. Competition EMSA with “positive” oligomer pairs from APOE, APP, BACE1 and TP53 promoters vs. Aβ1–42 peptide

A.

Aβ1–42 peptide was incubated with radiolabeled oligomers that contained the putative AβIDs in the APOE (lanes 1–4) 5’–flanking region at positions +171 (lane 1), −899 (lane 2), +284 (lane 3), +660/+665 (lane 4); in the APP (lanes 5–8) 5’–flanking region at positions −1862 (lane 5), −2871 (lane 6), −3364 (lane 7), and −3833 (lane 8); and in the BACE1 (lanes 9–12) 5’–flanking region at positions −119 (lane 9), −1766 (lane 10), −1939 (lane 11), and +36 (lane 12). In addition, an oligomer containing the Aβ–binding element from p53 was labeled and run (lane 13). The gel was dried and exposed to X–ray film for autoradiography. Specific oligomer pairs that were selected for competition EMSA are indicated with “*”.

B.

Aβ1–42 was incubated with radiolabeled oligomers that had previously shown interaction with Aβ1–42 (lanes 1,3,5,7,9,11,13,15). These reactions were repeated with the addition of 140× molar excess unlabeled oligomer (lanes 2,4,6,8,10,12,14,16). Specificity of Aβ/oligomer interaction is demonstrated by a significant reduction of autoradiograph signal at top of lane. The gel was dried and exposed to X–ray film for autoradiography. DNA–peptide interactions appeared as dark signal near the top of the gel.

Figure 5

Figure 5. Structure of the Aβ–binding domain (AβID) consensus motif

Combined height of stacked letters corresponds to bits of information (Shannon, 1997). An asterisk indicates the position of a “G” that may be critical for Aβ binding activity.

Figure 6

Figure 6. EMSA of different fragments of the Aβ peptide vs. four different DNA probes

Fragments of Aβ peptide corresponding to residues 1–42, 1–40, 1–28, 20–29, 25–35, 29–40, and 31–35 were self–oligomerized. In addition, “reverse” fragments 42–1, 40–1, and 35–25 were prepared.

A.

Fragments were incubated against oligomers the contained the Aβ–binding sequence at APOE +171 (lanes 1–10) or APOE +660 (lanes 11–20). The gel was dried and exposed to X–ray film for autoradiography. DNA–peptide interactions appeared as dark signal near the top of the gel.

B.

Fragments were incubated against oligomers the contained the Aβ–binding sequence at APP −3833G (lanes 1–10) or APP −3833A (with a “G”→“A” substitution) (lanes 11–20). The gel was dried and exposed to X–ray film for autoradiography. DNA–peptide interactions appeared as dark signal near the top of the gel.

Figure 7

Figure 7. Sequences and alignment of different Aβ fragments used in EMSA

All diagrams are aligned and to the same scale.

A.

“Helical–form” secondary structure of Aβ1–42 in aqueous environment (Tomaselli et al., 2006), helices indicated by cylinders.

B.

Secondary structure of Aβ1–42 in apolar solvent (Crescenzi et al., 2002), helices indicated by cylinders.

C.

Forward sequences. Sequences for Aβ 1–42, 1–40, 1–28, 20–29, 25–35, 29–40, and 31–35 are depicted as aligned amino acid residues. Those peptides with no apparent DNA–binding activity are in gray background.

D.

Reverse sequences. Sequences for Aβ42–1, 40–1, and 35–25 are depicted as aligned amino acid residues. Those peptides with no apparent DNA–binding activity are in gray background.

Figure 8

Figure 8. Concentration dependency of Aβ–DNA interaction

A.

The oligomer containing the Aβ–binding consensus at APP −3833G was incubated with fragments of the Aβ peptide, specifically Aβ1–40 (lanes 2–6), 1–42 (lanes 7–10), 42–1 (lanes 11–13), 1–28 (lanes 14–16), and 25–35 (lanes 17–20) at different peptide concentrations as indicated. The gel was dried and exposed to X–ray film for autoradiography. DNA–peptide interactions appeared as dark signal near top of gel.

B.

The oligomers containing the Aβ–binding consensus at APOE +660 (lanes 1–10) and at BACE1 −119 (lanes 11–20) were incubated with fragments of the Aβ peptide, specifically Aβ1–40 (lanes 1–2, 11–12), 1–42 (lanes 3–4, 13–14), 42–1 (lanes 5–6, 15–16), 1–28 (lanes 7–8, 17–18), and 25–35 (lanes 9–10, 19–20) at different peptide concentrations as indicated. The gel was dried and exposed to X–ray film for autoradiography. DNA–peptide interactions appeared as dark signal near top of gel.

Figure 9

Figure 9. Presence of confirmed AβIDs and putative motifs as predicted by dynamic weight scores on selected gene 5’−flanking regions

The 5’–flanking sequences of several genes were searched for decamers, using the weight matrix we generated. Matrix–matching sites are indicated on the sequences. Forward–orientation sites are above the sequence line. Reverse–orientation sites are below the sequence line. The 5’–flanking regions from 4kb upstream to 1 kb downstream of the +1 transcription start site were analyzed for the APOE, APP, BACE1, TP53, ASCL1, BACE2, IDE, MAPT, OLIG2, and SLC38A1 gene sequences. Positions that corresponded to oligomers for our EMSA herein, all positive on EMSA, are boldface. A site on the APP sequence marked with “§” crosses a previously–characterized APP polymorphism associated with late–onset familial AD.

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