Somatostatin, an in vivo binder to Aβ oligomers, Binds to βPFOAβ(1−42) Tetramers

Somatostatin (SST14) is strongly related to Alzheimer's disease (AD), as its levels decline during aging, it regulates the proteolytic degradation of the amyloid beta peptide (Aβ), and it binds to Aβ oligomers in vivo. Recently, the 3D structure of a membrane-associated β-sheet pore-forming tetramer (βPFOAβ(1−42) tetramer) has been reported. Here, we show that SST14 binds selectively to the βPFOAβ(1−42) tetramer with a KD value of ∼40 μM without binding to monomeric Aβ(1−42). Specific NMR chemical shift perturbations, observed during titration of SST14, define a binding site in the βPFOAβ(1−42) tetramer and are in agreement with a 2:1 stoichiometry determined by both native mass spectroscopy and isothermal titration calorimetry. These results enabled us to perform driven docking and model the binding mode for the interaction. The present study provides additional evidence on the relation between SST14 and the amyloid cascade and positions the βPFOAβ(1−42) tetramer as a relevant aggregation form of Aβ and as a potential target for AD

Alzheimer´s Disease-associated Aβ42 Peptide: Expression and Purification for NMR Structural Studies

Background: The aggregation of the amyloid-beta peptide (Aβ) in the brain is strongly associated with Alzheimer´s disease (AD). However, the heterogeneous and transient nature of this process has prevented identification of the exact molecular form of Aβ responsible for the neurotoxicity observed in this disease. Therefore, characterizing Aβ aggregation is of utmost importance in the field of AD. Nuclear magnetic resonance spectroscopy (NMR) is a technique that holds great potential to achieve this goal. However, it requires the use of specific labels introduced through recombinant expression of Aβ. Objective: In this paper, we report on a straightforward expression and purification protocol to obtain [U-15N] and [U-2H,13C,15N] Aβ42. Method: Aβ42 is expressed fused to Small Ubiquitin-like Modifier (SUMO) protein, which prevents Aβ42 aggregation. Results: The solubilizing capacity of SUMO has allowed us to design a purification protocol involving immobilized metal affinity chromatography (IMAC), a desalting step, and two size exclusion chromatography (SEC) purifications. Conclusion: This approach, which does not require the use of costly and time-consuming reversed phase high performance liquid chromatography (RP-HPLC), offers a much straightforward strategy to those previously described to obtain [U-15N] Aβ42 and it is the first protocol through which to achieve [U-2H,13C,15N] Aβ42. The peptides obtained are of high purity and have the required isotope enrichment to support NMR-based structural studies

Direct evidence of the presence of cross-linked Aβ dimers in the brains of Alzheimer's disease patients

Brain-derived amyloid-β (Aβ) dimers are associated with Alzheimer´s disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link would make brain-derived dimers (brain dimers) more synaptotoxic than Aβ monomers and would make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aβ (CL Aβ) dimers, obtained by means of the photo-induced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative brain CL Aβ dimers. Second, we used these PICUP CL Aβ dimers as standards to improve the isolation of brain Aβ dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of brain Aβ dimer samples allowing the detection of the CL [Aβ(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aβ dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease

Alzheimer´s Disease-associated Aβ42 Peptide: Expression and Purification for NMR Structural Studies

Background: The aggregation of the amyloid-beta peptide (Aβ) in the brain is strongly associated with Alzheimer´s disease (AD). However, the heterogeneous and transient nature of this process has prevented identification of the exact molecular form of Aβ responsible for the neurotoxicity observed in this disease. Therefore, characterizing Aβ aggregation is of utmost importance in the field of AD. Nuclear magnetic resonance spectroscopy (NMR) is a technique that holds great potential to achieve this goal. However, it requires the use of specific labels introduced through recombinant expression of Aβ. Objective: In this paper, we report on a straightforward expression and purification protocol to obtain [U-15N] and [U-2H,13C,15N] Aβ42. Method: Aβ42 is expressed fused to Small Ubiquitin-like Modifier (SUMO) protein, which prevents Aβ42 aggregation. Results: The solubilizing capacity of SUMO has allowed us to design a purification protocol involving immobilized metal affinity chromatography (IMAC), a desalting step, and two size exclusion chromatography (SEC) purifications. Conclusion: This approach, which does not require the use of costly and time-consuming reversed phase high performance liquid chromatography (RP-HPLC), offers a much straightforward strategy to those previously described to obtain [U-15N] Aβ42 and it is the first protocol through which to achieve [U-2H,13C,15N] Aβ42. The peptides obtained are of high purity and have the required isotope enrichment to support NMR-based structural studies

Direct evidence of the presence of cross-linked Aβ dimers in the brains of Alzheimer's disease patients

Brain-derived amyloid-β (Aβ) dimers are associated with Alzheimer´s disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link would make brain-derived dimers (brain dimers) more synaptotoxic than Aβ monomers and would make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aβ (CL Aβ) dimers, obtained by means of the photo-induced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative brain CL Aβ dimers. Second, we used these PICUP CL Aβ dimers as standards to improve the isolation of brain Aβ dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of brain Aβ dimer samples allowing the detection of the CL [Aβ(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aβ dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease