Mechanisms behind the Fibrillation and Toxicity of Insulin Fibrils on Neuron Cells by Engineered Curcumin Analogs

Abstract

Among foods, the use of plant derivatives as promising drugs and/or excipients has been considered from various perspectives. In the present study, curcumin, which is one of the most important plant derivatives for biological uses, and four curcumin-based pyrido­[2,3-d]­pyrimidine analogs (C2-C5) were used for investigating the mechanism of insulin fibrillation and evaluating the cytotoxicity of insulin fibrils. The synthesized analogs differed in terms of hydrophobicity and electrostatic charge. The analogs with more hydrophobicity (C1 and C4) in both acidic and neutral environments were able to reduce the rate of insulin fibrillation and the degree of cross-linking in the produced fibrils. Additionally, the toxicity of these fibrils for neural cells (N2a cell line) was very low. However, they did not show any significant effects on the toxicity of non-neural cells (HEK293 cell line), indicating the effect of the biochemical surface diversity on determining the vulnerability to fibrils and even the mechanism of action of additives on cell line survival. Although negatively charged analogs were able to reduce insulin fibrillation in the acidic environment, they indicated an opposite effect in the neutral environment. The resultant fibrils in the acidic medium appeared with a well-distinguished filament, but they were very close at neutral pH levels. Moreover, such fibrils indicated very poor toxicity against the N2a cell line and had no significant effects on HEK293 cells. Considering the docking studies, by creatively using the size exclusion chromatography, it was suggested that analogs C2 and C3 were capable of binding to the C-terminal end of the insulin B chain (low affinity) and HisB10 (high affinity). Hence, it was suggested that different compounds could play different protecting and/or destroying roles in cell toxicity by blocking some ligands at the surface of neuron cells.

Fabricating Multiphasic Angiogenic Scaffolds Using Amyloid/Roxadustat-Assisted High-Temperature Protein Printing

“…Additionally, other studies have mentioned the binding of amyloids on cells by their electrostatic charge. , The positive charge present in amyloids may lead to their better binding to cells with a negative charge, but this mechanism does not seem to be universal; in the present study, despite the negative charge of HSA-F (Figure ), we witnessed the strong attachment of cells to the scaffold (Figures and ). However, in a study that we had done before, we found that the positive charge of the monomers that make amyloids can lead to a delay in their fibrillation process and general structure . The lingering question pertains to what constituents present on the surface of amyloids evoke favorable responses from cells.…”

“…Therefore, to measure the thermal stability of HSA-F, the investigation of the secondary structures by ATR-FTIR in the dry state and Raman spectroscopy in the water-soluble state was used (Figure D). To investigate the secondary structures of proteins by FTIR spectra, the amide-II region, which covers the wavenumber range between 1600 and 1700 cm –1 , is of great importance. ,, Compared to the HSA monomer, which contains 29.5% α-helix structure, HSA-F has undergone α-helix to β-sheet transition to contain 40.3% β-sheet. After HSA-F was incubated at 140 °C for 3 h, it was seen that part of the helix and β-sheet turned into extended chain structures (Table S2).…”

“…To confirm the transformation of the globular state of HSA to the fibril state (HSA-F), Thioflavin T (ThT) was used. ThT is a fluorophore that binds to the β-sheets formed in amyloid fibrils and increases its λ max emission at a wavelength of 484 nm. , To prepare 1.0 mM ThT stock in double-distilled water, the molar extinction coefficient of 24,420 M –1 cm –1 at 420 nm was used . To confirm fibril formation, 0.2 mg/mL of HSA-F was prepared in tris buffer (20 mM, pH 7.4), and ThT was added with a final concentration of 10 μM.…”

“…In our previous study, we observed that the hydrophobic connection between insulin amyloids and N2a nerve cells, which are rich in ganglioside GM1, can be a bridge for the toxicity of insulin fibril to the cells and a precursor to the connection between diabetes and Alzheimer's disease. 50 In addition to helping to attach the cell to the scaffold (Figure 3), it appears that amyloids can also increase cell migration, giving rise to the theory that amyloids may serve as highways for cell movement. 84 This increase in cell migration may be another mechanism for increasing angiogenesis in Figures 9 and 11, as one of the pathways that VEGF initiates in HUVEC is to increase angiogenesis by increasing migration.…”

“…However, in a study that we had done before, we found that the positive charge of the monomers that make amyloids can lead to a delay in their fibrillation process and general structure. 50 The lingering question pertains to what constituents present on the surface of amyloids evoke favorable responses from cells. The current consensus regarding the mechanism of cytotoxicity and the adhesive properties of amyloids suggest that these two processes operate via distinct mechanisms, and engaging in one does not inherently precipitate the other.…”