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Butyrylcholinesterase interactions with amylin may protect pancreatic cells in metabolic syndrome.

Shenhar-Tsarfaty S, Bruck T, Bennett ER, Bravman T, Aassayag EB, Waiskopf N, Rogowski O, Bornstein N, Berliner S, Soreq H - J. Cell. Mol. Med. (2011)

Bottom Line: However, the activity differences remained unexplained.We demonstrate that BChE interacts with amylin through its core domain and efficiently attenuates both amylin fibril and oligomer formation.Taken together, our results suggest that MetS-associated BChE increases could protect pancreatic β-cells in vivo by decreasing the formation of toxic amylin oligomers.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Internal Medicine, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

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Direct rhBChE-amylin interactions. (A) Cartoon of the human BChE dimer based on a 2.8 Å X-ray structure with each monomer coloured from blue at the N-terminus to red at the C-terminus [50]. (B) Sequence of C-terminal peptides BSP-U and BSP-K with the A6T change marked in blue. (C) Immunoblot of amylin-BChE complex. Amylin and rhBChE were incubated and the soluble fraction cross-linked as described in ‘Materials and methods’. The complex was separated by 8% SDS-PAGE, transferred to nitrocellulose, and amylin was detected using a specific primary antibody, horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. The positions of the molecular mass standards are shown at the left. (D) SPR sensorgram showing dose-dependent amylin-BChE interactions. rhBChE was immobilized to a GLC chip and five increasing concentrations of amylin (red, 4.7 μM; green, 9.5 μM; blue, 19 μM; cyan, 38 μM; pink, 75.8 μM) were injected over the chip, in addition to running buffer (yellow). Background (interaction of injected amylin with IgG) was subtracted from all the traces shown. (E) Sensorgram showing dose-dependent interactions between amylin immobilized to a GLC chip and amylin injected over the chip. (F) Sensorgram showing lack of interaction between immobilized BSP-U and injected amylin: all traces are at background RU and show no dose-dependence. (G) Sensorgram showing lack of interaction between immobilized BSP-K and injected amylin. (H) Sensorgram showing dose-dependent interactions between immobilized AChE-R and injected amylin. All experiments were carried out at least twice on different days, using different protein and peptide solutions, and the traces shown are representative.
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fig03: Direct rhBChE-amylin interactions. (A) Cartoon of the human BChE dimer based on a 2.8 Å X-ray structure with each monomer coloured from blue at the N-terminus to red at the C-terminus [50]. (B) Sequence of C-terminal peptides BSP-U and BSP-K with the A6T change marked in blue. (C) Immunoblot of amylin-BChE complex. Amylin and rhBChE were incubated and the soluble fraction cross-linked as described in ‘Materials and methods’. The complex was separated by 8% SDS-PAGE, transferred to nitrocellulose, and amylin was detected using a specific primary antibody, horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. The positions of the molecular mass standards are shown at the left. (D) SPR sensorgram showing dose-dependent amylin-BChE interactions. rhBChE was immobilized to a GLC chip and five increasing concentrations of amylin (red, 4.7 μM; green, 9.5 μM; blue, 19 μM; cyan, 38 μM; pink, 75.8 μM) were injected over the chip, in addition to running buffer (yellow). Background (interaction of injected amylin with IgG) was subtracted from all the traces shown. (E) Sensorgram showing dose-dependent interactions between amylin immobilized to a GLC chip and amylin injected over the chip. (F) Sensorgram showing lack of interaction between immobilized BSP-U and injected amylin: all traces are at background RU and show no dose-dependence. (G) Sensorgram showing lack of interaction between immobilized BSP-K and injected amylin. (H) Sensorgram showing dose-dependent interactions between immobilized AChE-R and injected amylin. All experiments were carried out at least twice on different days, using different protein and peptide solutions, and the traces shown are representative.

Mentions: To examine the involvement of BChE protein–peptide interactions in the shift from MetS to T2DM, we performed PICUP of highly purified rhBChE of the U variant (Fig. 3A) with the amylin peptide. This method enables the covalent cross-linking of proteins or peptides found in close proximity, without the use of modifications or chemical cross-linkers. Following incubation and cross-linking, the resultant complexes of rBChE and amylin were separated by gel electrophoresis and immune-stained for amylin. A pronounced double band of over 250 kD is seen (Fig. 3C), representing rhBChE tetramers (∼280 kD) complexed with amylin (the doublet may represent two distinct forms of these complexes), as well as an amylin hexamer band (∼27 kD). Samples containing rhBChE alone showed no staining for amylin.


Butyrylcholinesterase interactions with amylin may protect pancreatic cells in metabolic syndrome.

Shenhar-Tsarfaty S, Bruck T, Bennett ER, Bravman T, Aassayag EB, Waiskopf N, Rogowski O, Bornstein N, Berliner S, Soreq H - J. Cell. Mol. Med. (2011)

Direct rhBChE-amylin interactions. (A) Cartoon of the human BChE dimer based on a 2.8 Å X-ray structure with each monomer coloured from blue at the N-terminus to red at the C-terminus [50]. (B) Sequence of C-terminal peptides BSP-U and BSP-K with the A6T change marked in blue. (C) Immunoblot of amylin-BChE complex. Amylin and rhBChE were incubated and the soluble fraction cross-linked as described in ‘Materials and methods’. The complex was separated by 8% SDS-PAGE, transferred to nitrocellulose, and amylin was detected using a specific primary antibody, horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. The positions of the molecular mass standards are shown at the left. (D) SPR sensorgram showing dose-dependent amylin-BChE interactions. rhBChE was immobilized to a GLC chip and five increasing concentrations of amylin (red, 4.7 μM; green, 9.5 μM; blue, 19 μM; cyan, 38 μM; pink, 75.8 μM) were injected over the chip, in addition to running buffer (yellow). Background (interaction of injected amylin with IgG) was subtracted from all the traces shown. (E) Sensorgram showing dose-dependent interactions between amylin immobilized to a GLC chip and amylin injected over the chip. (F) Sensorgram showing lack of interaction between immobilized BSP-U and injected amylin: all traces are at background RU and show no dose-dependence. (G) Sensorgram showing lack of interaction between immobilized BSP-K and injected amylin. (H) Sensorgram showing dose-dependent interactions between immobilized AChE-R and injected amylin. All experiments were carried out at least twice on different days, using different protein and peptide solutions, and the traces shown are representative.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4373355&req=5

fig03: Direct rhBChE-amylin interactions. (A) Cartoon of the human BChE dimer based on a 2.8 Å X-ray structure with each monomer coloured from blue at the N-terminus to red at the C-terminus [50]. (B) Sequence of C-terminal peptides BSP-U and BSP-K with the A6T change marked in blue. (C) Immunoblot of amylin-BChE complex. Amylin and rhBChE were incubated and the soluble fraction cross-linked as described in ‘Materials and methods’. The complex was separated by 8% SDS-PAGE, transferred to nitrocellulose, and amylin was detected using a specific primary antibody, horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. The positions of the molecular mass standards are shown at the left. (D) SPR sensorgram showing dose-dependent amylin-BChE interactions. rhBChE was immobilized to a GLC chip and five increasing concentrations of amylin (red, 4.7 μM; green, 9.5 μM; blue, 19 μM; cyan, 38 μM; pink, 75.8 μM) were injected over the chip, in addition to running buffer (yellow). Background (interaction of injected amylin with IgG) was subtracted from all the traces shown. (E) Sensorgram showing dose-dependent interactions between amylin immobilized to a GLC chip and amylin injected over the chip. (F) Sensorgram showing lack of interaction between immobilized BSP-U and injected amylin: all traces are at background RU and show no dose-dependence. (G) Sensorgram showing lack of interaction between immobilized BSP-K and injected amylin. (H) Sensorgram showing dose-dependent interactions between immobilized AChE-R and injected amylin. All experiments were carried out at least twice on different days, using different protein and peptide solutions, and the traces shown are representative.
Mentions: To examine the involvement of BChE protein–peptide interactions in the shift from MetS to T2DM, we performed PICUP of highly purified rhBChE of the U variant (Fig. 3A) with the amylin peptide. This method enables the covalent cross-linking of proteins or peptides found in close proximity, without the use of modifications or chemical cross-linkers. Following incubation and cross-linking, the resultant complexes of rBChE and amylin were separated by gel electrophoresis and immune-stained for amylin. A pronounced double band of over 250 kD is seen (Fig. 3C), representing rhBChE tetramers (∼280 kD) complexed with amylin (the doublet may represent two distinct forms of these complexes), as well as an amylin hexamer band (∼27 kD). Samples containing rhBChE alone showed no staining for amylin.

Bottom Line: However, the activity differences remained unexplained.We demonstrate that BChE interacts with amylin through its core domain and efficiently attenuates both amylin fibril and oligomer formation.Taken together, our results suggest that MetS-associated BChE increases could protect pancreatic β-cells in vivo by decreasing the formation of toxic amylin oligomers.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Internal Medicine, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Show MeSH
Related in: MedlinePlus