Interaction of amyloid beta with humanin and acetylcholinesterase is modulated by ATP

Abstract

Humanin (HN) is known to bind amyloid beta (Aβ)-inducing cytoprotective effects, while binding of acetylcholinesterase (AChE) to Aβ increases its aggregation and cytotoxicity. Previously, we showed that binding of HN to Aβ blocks aggregation induced by AChE and that HN decreases but does not abolish Aβ-AChE interactions in A549 cell media. Here, we set out to shed light on factors that modulate the interactions of Aβ with HN and AChE. We found that binding of either HN or AChE to Aβ is not affected by heparan sulfate, while ATP, thought to reduce misfolding of Aβ, weakened interactions between AChE and Aβ but strengthened those between Aβ and HN. Using media from either A549 or H1299 lung cancer cells, we observed that more HN was bound to Aβ upon addition of ATP, while levels of AChE in a complex with Aβ were decreased by ATP addition to A549 cell media. Exogenous addition of ATP to either A549 or H1299 cell media increased interactions of endogenous HN with Aβ to a comparable extent despite differences in AChE expression in the two cell lines, and this was correlated with decreased binding of exogenously added HN to Aβ. Treatment with exogenous ATP had no effect on cell viability under all conditions examined. Exogenously added ATP did not affect viability of cells treated with AChE-immunodepleted media, and there was no apparent protection against the cytotoxicity resulting from immunodepletion of HN. Moreover, exogenously added ATP had no effect on the relative abundance of oligomer versus total Aβ in either cell line.

Keywords: acetylcholinesterase; amyloid-beta; humanin; kinetics; lung cancer; peptide interaction.

Fig. 1

(A) Amino acid sequence of Aβ40 and Aβ42. (B) Regions of Aβ known to bind AChE (1–16, 12–28, blue lines) and HN (17–28, brown lines) are shown along with the heparan sulfate and GxxxG motifs. Tyr10 and Ser26 of Aβ previously reported to interact with ATP are shown in blue highlights.

Fig. 2

HS does not alter the binding of Aβ to either HN or AChE. (A) HS (100 nm) was bound to ELISA plate wells. Increasing concentrations of biotinylated‐Aβ were added to the wells and processed as described in Materials and methods. Optical densities (450 nm) were normalized for both curves by expressing each point relative to the best‐fitted E _max value (set to 100%). The data were then plotted as a function of increasing biotinylated‐Aβ concentrations and, using the graphpad prism 8.4.3 software, fit to a single binding site model with a nonlinear regression curve fitting approach. Data were expressed as the mean ± SD of three independent experiments, each carried out in triplicate. (B) Aβ (100 nm) was bound to ELISA plate wells. Biotinylated‐HN (300 nm) was then added to the wells in the absence or presence of increasing concentrations of HS. (C) AChE (10 nm) was bound to the wells. Biotinylated‐Aβ (1 μm) was then added in the absence or presence of increasing concentrations of HS. The negative controls had the same HN or Aβ and AChE concentrations, but water was substituted in place of biotinylated‐Aβ or biotinylated‐HN. Data were processed using the graphpad prism 8.4.3 software and presented as the mean ± SD of three independent assays.

Fig. 3

ATP diminishes the binding of AChE to Aβ. AChE (10 nm) was bound to the wells and then increasing concentrations of biotinylated‐Aβ40 (A) or biotinylated‐Aβ42 (B), in the absence or presence of 200 μm ATP, were added to the wells and processed as described in Materials and methods. Optical density measurements (450 nm) were normalized by expressing each point in relation to the best‐fitted E _max value (set to 100%) and plotted as a function of increasing biotinylated‐Aβ concentrations. The data were fit to a single binding site model with a nonlinear regression curve fitting approach using graphpad prism 8.4.3. Data were expressed as the mean ± SD of three independent experiments, each run in triplicate.

Fig. 4

ATP increases the binding of HN to Aβ. Aβ40 (A, 100 nm) or Aβ42 (B, 100 nm), preincubated in a buffer in the absence or presence of 200 μm ATP, was bound to the wells, and then increasing concentrations of biotinylated‐HN were added and processed as described in Materials and methods. Optical density measurements (450 nm) were normalized by expressing each point in relation to the best‐fitted E _max value (set to 100%) and then plotted as a function of increasing biotinylated‐HN concentrations. The data were fit to a single binding site model with a nonlinear regression curve fitting approach using graphpad prism 8.4.3. Data were expressed as the mean ± SD of three independent experiments, each performed in triplicate.

Fig. 5

ATP increases the amount of HN found in a complex with Aβ in both A549 and H1299 cell media while AChE found in a complex with Aβ is decreased by the addition of ATP to A549 cell media. Cells (0.2 × 10⁵ cells per well) were seeded in 96‐well plates in 10% FBS‐supplemented media. The next day, the cells were incubated in serum‐free medium for 72 h. Specific antibodies were added (1 : 1000 dilution) to ELISA wells. After blocking the wells, 300 μL of the A549 (A, B) or H1299 (C, D) cell‐conditioned medium (0.5 μg·μL⁻¹), 72 h postserum starvation, was added. The proteins/peptides were detected using their corresponding primary antibodies and then processed as described in Materials and methods section. Each column represents the mean ± SD of three independent separate experiments, each performed in triplicate. Data processing was carried out using the graphpad 8.4.3 software. Asterisks (**) indicate a statistically significant difference between each treatment relative to samples without ATP. Absence of asterisks indicates no significance, Mann–Whitney test, **P < 0.01.

Fig. 6

Exogenously added ATP increases HN bound to Aβ from media of A549 and H1299 cells using 82E1 antibodies, resulting in decreased binding of exogenously added HN to Aβ. Conversely, less AChE is found in a complex with Aβ in the presence of added ATP in A549 cells. Anti‐Aβ‐specific antibodies (82E1) were added (1 : 1000 dilution) to ELISA plate wells. The wells were blocked, and 300 µL of conditioned media (0.5 µg·µL⁻¹) of A549 cells (A) or H1299 cells (B), 72 h after serum starvation, was added without or with ATP and increasing HN concentrations, and then, the HN and AChE bound were detected using the corresponding specific primary antibodies and processed as described in Materials and methods. Fold change relative to controls incubated with all components except the primary antibodies was calculated and fit, using the graphpad prism 8.4.3 software, with a nonlinear regression curve. The data represent the mean ± SD of three separate experiments, each performed in triplicate.

Fig. 7

Addition of exogenous ATP has no effect on A549 or H1299 cell viability. Viability of A549 (A) or H1299 (B) cells was assessed by the MTT assay. Cells were seeded in 96‐well plates at 0.2 × 10⁵ cells per well in 200 µL 10% FBS‐supplemented media. The next day, the cell monolayers were incubated in serum‐free medium for 12 h, then treated with control media, HN or AChE‐ID media (Media ID), without or with ATP, for 48 h with the media containing the specific components in the various treatments replaced every 12 h. Data were processed using the graphpad 8.4.3 software. The graphs summarize the results expressed as means ± SD of three separate experiments, each performed in triplicate. Statistical differences between ID versus nondepleted media were analyzed by a one‐way analysis of variance (ANOVA) test, **P < 0.01.

Fig. 8

Addition of ATP does not affect the relative amount of oligomer versus total Aβ upon ID of HN from A549 (A) or H1299 (B) cell‐conditioned media. Cells (0.2 × 10⁵) were grown in 10% FBS‐supplemented media for 24 h. The cells were then incubated in serum‐free medium for 72 h without or with ATP and the media collected and ID from either HN or AChE as described in Materials and methods section. The antibodies 6E10 or 4G8 were bound (1 : 1000 dilution) to ELISA wells. The wells were blocked, and then incubated with 300 μL of the control and ID medium (0.5 μg·μL⁻¹). Biotin‐4G8 was then added and the signal processed as described in Materials and methods section. Fold change relative to controls using anti‐6E10 and anti‐4G8 antibodies and 300 μL of the medium not incubated with cells was calculated. Data were processed using the graphpad 8.4.3 software. The graphs summarize the results expressed as means ± SD of three separate experiments, each performed in triplicate. Absence of asterisks indicates no significance compared to samples without added ATP, Mann–Whitney test.

Fig. 9

A graphic representation of the key findings of the current investigation. Both HN and AChE are able to bind Aβ in the absence of added ATP. Addition of ATP increases the binding affinity of Aβ to HN but not to AChE.