Protein disulfide isomerase (PDI) is mainly located in the endoplasmic reticulum (ER) but is also secreted into the bloodstream where its oxidoreductase activity is involved with thrombus formation. Quercetin-3-rutinoside (Q3R) blocks this activity, but its inhibitory mechanism against PDI is not fully understood. Here, we examined the potential inhibitory effect of Q3R on another process that requires PDI: disassembly of the multimeric cholera toxin (CT). In the ER, PDI physically displaces the reduced CTA1 subunit from its non-covalent assembly in the CT holotoxin. This is followed by CTA1 dislocation from the ER to the cytosol where the toxin interacts with its G protein target for a cytopathic effect. Q3R blocked the conformational change in PDI that accompanies its binding to CTA1, which, in turn, prevented PDI from displacing CTA1 from its holotoxin and generated a toxin-resistant phenotype. Other steps of the CT intoxication process were not affected by Q3R, including PDI binding to CTA1 and CT reduction by PDI. Additional experiments with the B chain of ricin toxin found that Q3R could also disrupt PDI function through the loss of substrate binding. Q3R can thus inhibit PDI function through distinct mechanisms in a substrate-dependent manner.
Amyloid β (Aβ) peptide aggregation plays a central role in Alzheimer’s disease (AD) etiology. AD drug candidates have included small molecules or peptides directed towards inhibition of Aβ fibrillogenesis. Although some Aβ-derived peptide fragments suppress Aβ fibril growth, comprehensive analysis of inhibitory potencies of peptide fragments along the whole Aβ sequence has not been reported. The aim of this work is (a) to identify the region(s) of Aβ with highest propensities for aggregation and (b) to use those fragments to inhibit Aβ fibrillogenesis. Structural and aggregation properties of the parent Aβ1–42 peptide and seven overlapping peptide fragments have been studied, i.e. Aβ1–10 (P1), Aβ6–15 (P2), Aβ11–20 (P3), Aβ16–25 (P4), Aβ21–30 (P5), Aβ26–36 (P6), and Aβ31–42 (P7). Structural transitions of the peptides in aqueous buffer have been monitored by circular dichroism and Fourier transform infrared spectroscopy. Aggregation and fibrillogenesis were analyzed by light scattering and thioflavin-T fluorescence. The mode of peptide-peptide interactions was characterized by fluorescence resonance energy transfer. Three peptide fragments, P3, P6, and P7, exhibited exceptionally high propensity for β-sheet formation and aggregation. Remarkably, only P3 and P6 exerted strong inhibitory effect on the aggregation of Aβ1–42, whereas P7 and P2 displayed moderate inhibitory potency. It is proposed that P3 and P6 intercalate between Aβ1–42 molecules and thereby inhibit Aβ1–42 aggregation. These findings may facilitate therapeutic strategies of inhibition of Aβ fibrillogenesis by Aβ-derived peptides.
Amyloid β (Aβ) peptide aggregates are linked to Alzheimer's disease (AD). Posttranslationally pyroglutamylated Aβ (pEAβ) occurs in AD brains in significant quantities and is hypertoxic, but the underlying structural and aggregation properties remain poorly understood. Here, the structure and aggregation of Aβ1–40 and pEAβ3–40 are analyzed separately and in equimolar combination. Circular dichroism data show that Aβ1–40, pEAβ3–40, and their combination assume α‐helical structure in dry state and transition to unordered structure in aqueous buffer. Aβ1–40 and the 1:1 combination gradually acquire β‐sheet structure while pEAβ3–40 adopts an α‐helix/β‐sheet conformation. Thioflavin‐T fluorescence studies suggest that the two peptides mutually inhibit fibrillogenesis. Fourier transform infrared (FTIR) spectroscopy identifies the presence of β‐turn and α‐helical structures in addition to β‐sheet structure in peptides in aqueous buffer. The kinetics of transitions from the initial α‐helical structure to β‐sheet structure were resolved by slow hydration of dry peptides by D2O vapor, coupled with isotope‐edited FTIR. These data confirmed the mutual suppression of β‐sheet formation by the two peptides. Remarkably, pEAβ3–40 maintained a significant fraction of α‐helical structure in the combined sample, implying a reduced β‐sheet propensity of pEAβ3–40. Altogether, the data imply that the combination of unmodified and pyroglutamylated Aβ peptides resists fibrillogenesis and favors the prefibrillar state, which may underlie hypertoxicity of pEAβ.
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