Brazilin inhibits fibrillogenesis of human islet amyloid polypeptide, disassembles mature fibrils, and alleviates cytotoxicity

Abstract

Fibrillogenesis of human islet amyloid polypeptide (hIAPP) is a pathological hallmark of type II diabetes mellitus (T2DM), and the inhibition of hIAPP fibrillogenesis is an important strategy for the prevention and treatment of T2DM. In this study, the inhibitory effects of brazilin on the fibrillization and cytotoxicity of hIAPP were examined using the thioflavin T fluorescence (ThT) assay, transmission electron microscopy (TEM), circular dichroism (CD) spectroscopy, cytotoxicity assays, and molecular dynamics simulations.Both the ThT and TEM results have shown that brazilin inhibits hIAPP fibrillogenesis in a dose-dependent manner. CD studies revealed that brazilin delays the conformational transition of hIAPP from its initial ahelical to the b-sheet form. As a result, brazilin greatly alleviates hIAPP-induced cytotoxicity. Moreover, we also found that brazilin disassembles preexisting hIAPP fibrils, and alleviates the cytotoxicity of hIAPP aggregates. The results of free energy decomposition studies calculated using molecular mechanicsPoisson-Boltzmann surface area analysis revealed that hydrophobic interactions contribute more than 75% of the free energy of binding in the brazilin-hIAPP complex, while electrostatic interactions (i.e., hydrogen bonds) play a secondary role (<25%). Two binding sites of brazilin on the hIAPP pentamer were identified, encompassing the N-terminal region and the turn region. There are 11 important residues of hIAPP that strongly interact with brazilin -Asn3, Thr4, Thr9, Arg11, Asn14, Phe15, His18, Ser19, Ser20, Asn21 and Phe23. The findings presented here will contribute to a comprehensive understanding of the inhibitory effect of brazilin on the fibrillogenesis of hIAPP, which is critical for the search for more effective agents that can inhibit hIAPP fibrillogenesis.

His18 promotes reactive oxidative stress production in copper-ion mediated human islet amyloid polypeptide aggregation

“…The aggregation process of hIAPP has been proved to be modulated by many factors including pH value, metal ions, small chemical molecules, etc. [11][12][13][14][15] Copper ions are believed to be closely associated with T2D as clinical ndings show that the serum copper ions levels are higher in diabetic patients than in healthy people. 16,17 Recently, the particular role of copper ions in diseases associated with hIAPP aggregation has been studied extensively.…”

“…In order to clarify the mechanism of Cu(II) promoting the generation of ROS, in this study the interaction of Cu(II) with hIAPP was explored using different spectroscopy techniques. hIAPP (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) fragment was used as a main model, as it contains the region (20)(21)(22)(23)(24)(25)(26)(27)(28) for amyloid formation as well as the main binding site (His18) for the metal ions. 10,13,29 We rst compared the amount of H 2 O 2 production in several fragments co-incubated with copper ions including hIAPP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), hIAPP (11)(12)(13)(14)(15)(16)(17)…”

Multifaceted Influences of Melanin‐Like Particles on Amyloid‐beta Aggregation

“…amyloid-b and a-synuclein). [4][5][6][7] Many relevant studies using other polyphenols or catechols, such as baicalein, [8][9][10][11] taxifolin, [12] epicatechin, [13] and brazilin [14,15] for different amyloidogenicp roteins/peptides showed similarr esults. In particular, the role of dopamine-one of the simplest biologically occurring catechols-in amyloid aggregation andt he resultant pathology of Parkinson's disease( PD) has been an important subject of studies, [16,17] as PD occurs at ad opaminergic neuron-rich domain of ab rain, the substantia nigra, and as ignificant amount of dopamine is found in Lewy bodies-the hallmark proteinaceous aggregateso fP D. [18,19] Yetn atural polyphenols have been originally suspected, recentr esultsc ollectively suggest that (i)catecholi st he minimal molecular unit that can interfere with protein aggregation, (ii)its influences are general, with slightly different degrees, for many amyloidogenic proteins, and (iii)the O 2 -drivena uto-oxidation of catechols is somehow deeply involved in such influences.…”

Highly efficient soluble expression, purification and characterization of recombinant Aβ42 fromEscherichia coli

“…5A. When 25 mM recombinant Ab42 was incubated at 37 C for 72 h, the ThT uorescence prole showed an almost negligible lag phase, a fast growth phase within 20 h, and a steady equilibrium phase aer 24 h. Fig. 5B shows the AFM images of Ab42 aer 0 and 3 days of incubation, which conrmed that the recombinant Ab42 formed typical long, branched brils with lengths of 200-700 nm.…”

“…The ThT uorescence assay is considered a highly sensitive tool for detecting the formation of amyloid aggregates of various amyloidogenic proteins. [37][38][39] Therefore, this assay was used to characterize the aggregation kinetics of recombinant Ab42 as shown in Fig. 5A.…”