To avoid potential ambiguity in the structural determination of native spider silk, we prepared both a nonlabeled peptide with a sequence containing both the polyalanine and the repeated GGA regions, QGAG(A) 6GGAGA(GGA)3GAGRGGLGG (I), and the 13 C-labeled peptides QGAGAAA[1-as a local structural model of spidroin 1 (MaSpl) protein in spider dragline silk of Nephila clavipes. Solvent treatments prior to the NMR measurements induce a structural change of these model peptides and provide a model to reproduce the structure of the silk fiber. Conformation-dependent 13 C NMR chemical shifts were mainly used to determine the local structure, including the evaluation of the fraction of several conformations. The characteristic structure, 65% β-sheet for the Ala 8 residue in the poly-Ala region, and 70% 31-helix for the Ala 21 residue and mainly 31-helix for the Gly 20 residue in the GG 20 A 21 sequence was observed after peptides Ia and II were dissolved in 9 M LiBr followed by dialysis against water. The 2D spin diffusion 13 C solid-state NMR spectrum of the Ala 21 residue of peptide II after this treatment was also reproduced by 70% 31-helix (φ, φ ) -90°, 120°) and 30% β-sheet (φ, φ ) -150°, 150°) structure. However, the Ala Cβ peak assigned to the 31-helix in the spectrum of Ia is broad, implying that the torsion angles of the Ala 21 residue are distributed, but with an average that corresponds approximately to the torsion angles of the 31-helix. An increase in the fraction of β-sheet in both the poly-Ala and GG 20 A 21 regions was observed for Ia after it was dissolved in formic acid and then dried in air. Moreover, after Ia was dissolved in formic acid and then precipitated in methanol, the spectrum showed a tightly packed β-sheet structure with a further increase in the fraction of β-sheet although 15% 31-helix still remained in the GG 20 A 21 region. The β-sheet structure of the poly-Ala region and both 31-helix and β-sheet structures in the repeated GGA sequence are in agreement with the structural model for the native spider dragline silk fiber from N. clavipes from a previous NMR study ( van Beek,
The formation of soluble oligomers of amyloid β42 and 40 (Aβ42, Aβ40) is the initial event in the pathogenesis of Alzheimer's disease (AD). Based on previous systematic proline replacement and solid-state NMR, we proposed a toxic dimer structure of Aβ42, a highly aggregative alloform, with a turn at positions 22 and 23, and a hydrophobic core in the C-terminal region. However, in addition to Aβ42, Aβ40 dimers can also contribute to AD progression because of the more abundance of Aβ40 monomer in biological fluids. Here, we describe the synthesis and characterization of three dimer models of the toxic-conformation constrained E22P-Aβ40 using l,l-2,6-diaminopimeric acid (DAP) or l,l-2,8-diaminoazelaic acid (DAZ) linker at position 30, which is incorporated into the intermolecular parallel β-sheet region, and DAP at position 38 in the C-terminal hydrophobic core. E22P-A30DAP-Aβ40 dimer (1) and E22P-A30DAZ-Aβ40 dimer (2) existed mainly in oligomeric states even after 2 weeks incubation without forming fibrils, unlike the corresponding monomer. Their neurotoxicity toward SH-SY5Y neuroblastoma cells was very weak. In contrast, E22P-G38DAP-Aβ40 dimer (3) formed β-sheet-rich oligomeric aggregates, and exhibited more potent neurotoxicity than the corresponding monomer. Ion mobility-mass spectrometry suggested that high molecular-weight oligomers (12-24-mer) of 3 form, but not for 1 and 2 after 4 h incubation. These findings indicate that formation of the hydrophobic core at the C-terminus, rather than intermolecular parallel β-sheet, triggers the formation of toxic Aβ oligomers. Compound 3 may be a suitable model for studying the etiology of Alzheimer's disease.
Solid-state NMR is especially useful when the structures of peptides and proteins should be analyzed by taking into account the structural distribution, that is, the distribution of the torsion angle of the individual residue. In this study, two-dimensional spin-diffusion solid-state NMR spectra of 13C-double-labeled model peptides (GPGGA)6G of flagelliform silk were observed for studying the local structure in the solid state. The spin-diffusion NMR spectra calculated by assuming the torsion angles of the beta-spiral structure exclusively could not reproduce the observed spectra. In contrast, the spectra calculated by taking into account the statistical distribution of the torsion angles of the individual central residues in the sequences Ala-Gly-Pro, Gly-Pro-Gly, Pro-Gly-Gly, Gly-Gly-Ala, and Gly-Ala-Gly from PDB data could reproduce the observed spectra well. This indicates that the statistical distribution of the torsion angles should be considered for the structural model of (GPGGA)6G similar to the case of the model peptide of elastin.
A significant number of new chemical entities (NCEs) disappear due to cytochrome P450 (CYP)-mediated clinical drug-drug interactions in drug discovery. Therefore, a high throughput assay of CYP activities is necessary in order to evaluate the inhibitory or inducible potencies of CYP isoforms with NCEs in early drug discovery. Here, we developed and validated a high-throughput assay to simultaneously monitor the in vitro activities of 8 CYP isoforms. A cocktail of 9 probe substrates for the 8 major CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5) was incubated with human liver microsomes. Each substrate-derived metabolite was simultaneously analyzed by multiple reactions monitoring with a single ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run using stable isotope-labeled internal standards. The ultra-fast UPLC gradient allowed each metabolite to be separated within 1 min, providing quantitative linearity of over 2 orders of magnitude. CYP inhibition by 8 well-known inhibitors was confirmed by comparing single substrates with the substrate cocktail. The inhibition curve profiles and IC₅₀ values for all CYPs in the cocktail substrate were similar to those of single substrates. UPLC-MS/MS using a CYP substrate cocktail is a reliable and robust high-throughput method to accurately assess CYP inhibition potencies of newly developed drugs.
Herein we report that a preferable inhibition of the nucleation phase of Aβ42, related to the formation of toxic oligomers, by triterpenoids from medicinal herbs originates from a salt bridge of their carboxy groups with Lys16 and 28 in Aβ42. Such a direct interaction targeting the monomer, dimer, and trimer suppressed further oligomerization. In contrast, the corresponding congeners without carboxy groups failed to do so.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.