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Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

Liang H, Shi Y, Kou Z, Peng Y, Chen W, Li X, Li S, Wang Y, Wang F, Zhang X - PLoS ONE (2015)

Bottom Line: We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model.We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment.Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Psychiatric Disorders of Guangdong Province, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

ABSTRACT
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

No MeSH data available.


Related in: MedlinePlus

Selected aptamers to BACE1 exhibit good specificity and high affinity.(A-D) Binding curves of A1, A4, anti-BACE1 and an unrelated aptamer, U31. Mean absorbances for each aptamer concentration from three independent experiments were used to create the binding curve. (E) The specific interaction of aptamer A1 with BACE1 protein was identified via affinity purification on streptavidin beads of M17-APPsw cell lysate treated with biotin-labeled A1, followed by immunoblotting with anti-BACE1 antibody. A band at 70 kDa corresponded to the molecular weight of BACE1 and indicated that BACE1 was the specific target of the aptamer. Lane 1: A1 with M17-APPsw cell lysate. Lane 2: GP30 with M17-APPsw cell lysate. Lane 3: U31 with M17-APPsw cell lysate. Lane 4: blank control (no aptamer) group. Three independent experiments were performed.
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pone.0140733.g002: Selected aptamers to BACE1 exhibit good specificity and high affinity.(A-D) Binding curves of A1, A4, anti-BACE1 and an unrelated aptamer, U31. Mean absorbances for each aptamer concentration from three independent experiments were used to create the binding curve. (E) The specific interaction of aptamer A1 with BACE1 protein was identified via affinity purification on streptavidin beads of M17-APPsw cell lysate treated with biotin-labeled A1, followed by immunoblotting with anti-BACE1 antibody. A band at 70 kDa corresponded to the molecular weight of BACE1 and indicated that BACE1 was the specific target of the aptamer. Lane 1: A1 with M17-APPsw cell lysate. Lane 2: GP30 with M17-APPsw cell lysate. Lane 3: U31 with M17-APPsw cell lysate. Lane 4: blank control (no aptamer) group. Three independent experiments were performed.

Mentions: Indirect ELISA was performed to determine the binding affinity of the selected aptamers to BACE1. Anti-BACE1 antibody and an unrelated aptamer, U31, were used as the control. Similar to anti-BACE1, the binding curves of A1 and A4 to BACE1 fit well (Fig 2A–2D) with Kd values in the nanomolar range (Table 2). These findings indicate that A1 and A4 can specifically bind to BACE1 with high affinity. A1 had the highest repetition frequency and was subsequently selected for the pull-down assay, which illustrated that A1 specifically interacted with BACE1 protein in M17-APPsw cells, whereas Gp30 and U31 did not exhibit binding to BACE1 proteins (Fig 2E).


Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

Liang H, Shi Y, Kou Z, Peng Y, Chen W, Li X, Li S, Wang Y, Wang F, Zhang X - PLoS ONE (2015)

Selected aptamers to BACE1 exhibit good specificity and high affinity.(A-D) Binding curves of A1, A4, anti-BACE1 and an unrelated aptamer, U31. Mean absorbances for each aptamer concentration from three independent experiments were used to create the binding curve. (E) The specific interaction of aptamer A1 with BACE1 protein was identified via affinity purification on streptavidin beads of M17-APPsw cell lysate treated with biotin-labeled A1, followed by immunoblotting with anti-BACE1 antibody. A band at 70 kDa corresponded to the molecular weight of BACE1 and indicated that BACE1 was the specific target of the aptamer. Lane 1: A1 with M17-APPsw cell lysate. Lane 2: GP30 with M17-APPsw cell lysate. Lane 3: U31 with M17-APPsw cell lysate. Lane 4: blank control (no aptamer) group. Three independent experiments were performed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608735&req=5

pone.0140733.g002: Selected aptamers to BACE1 exhibit good specificity and high affinity.(A-D) Binding curves of A1, A4, anti-BACE1 and an unrelated aptamer, U31. Mean absorbances for each aptamer concentration from three independent experiments were used to create the binding curve. (E) The specific interaction of aptamer A1 with BACE1 protein was identified via affinity purification on streptavidin beads of M17-APPsw cell lysate treated with biotin-labeled A1, followed by immunoblotting with anti-BACE1 antibody. A band at 70 kDa corresponded to the molecular weight of BACE1 and indicated that BACE1 was the specific target of the aptamer. Lane 1: A1 with M17-APPsw cell lysate. Lane 2: GP30 with M17-APPsw cell lysate. Lane 3: U31 with M17-APPsw cell lysate. Lane 4: blank control (no aptamer) group. Three independent experiments were performed.
Mentions: Indirect ELISA was performed to determine the binding affinity of the selected aptamers to BACE1. Anti-BACE1 antibody and an unrelated aptamer, U31, were used as the control. Similar to anti-BACE1, the binding curves of A1 and A4 to BACE1 fit well (Fig 2A–2D) with Kd values in the nanomolar range (Table 2). These findings indicate that A1 and A4 can specifically bind to BACE1 with high affinity. A1 had the highest repetition frequency and was subsequently selected for the pull-down assay, which illustrated that A1 specifically interacted with BACE1 protein in M17-APPsw cells, whereas Gp30 and U31 did not exhibit binding to BACE1 proteins (Fig 2E).

Bottom Line: We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model.We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment.Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Psychiatric Disorders of Guangdong Province, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

ABSTRACT
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

No MeSH data available.


Related in: MedlinePlus