Alzheimer's disease (AD) is the most prevalent form of dementia, and aggregation of amyloid β-proteins (Aβ) into soluble oligomers and fibrils has been implicated in the pathogenesis of AD. Herein we developed acidulated serum albumin for the inhibition of Aβ42 fibrillogenesis. Bovine serum albumin (BSA) was modified with diglycolic anhydride, leading to the coupling of 14.5 more negative charges (carboxyl groups) on average on each protein surface. The acidulated BSA (A-BSA) was characterized and confirmed to keep the tertiary structure and stability of BSA. Extensive biophysical and biological analyses showed that A-BSA significantly inhibited Aβ42 fibrillogenesis and mitigated amyloid cytotoxicity. As compared to the Aβ42-treated group (cell viability, 50%), the cell viability increased to 88% by the addition of equimolar A-BSA. The inhibitory effect was remarkably higher than that of BSA at the same concentration. On the basis of the experimental findings, a mechanistic model was proposed. The model considers that Aβ42 is bound to the A-BSA surface by hydrophobic interactions, but the widely distributed negative charges on the A-BSA surface give rise to electrostatic repulsions to the bound Aβ42 that is also negatively charged. The two well-balanced opposite forces make Aβ42 adopt extended conformations instead of the β-sheet structure that is necessary for the on-pathway fibrillogenesis, even when the protein is released off the surface. Thus, A-BSA greatly slows down the fibrillation and changes the fibrillogenesis pathway, leading to the formation of less toxic aggregates. The findings and the mechanistic model offer new insights into the development of more potent inhibitors of Aβ fibrillogenesis and cytotoxicity.

Fibrillogenesis of amyloid β-proteins (Aβ) mediated by transition-metal ions such as Zn(2+) in neuronal cells plays a causative role in Alzheimer's disease. Hence, it is highly desired to design multifunctional agents capable of inhibiting Aβ aggregation and modulating metal-Aβ species. In this study, we fabricated acidulated human serum albumin (A-HSA) as a multifunctional agent for binding Zn(2+) and modulating Zn(2+)-mediated Aβ fibrillogenesis and cytotoxicity. On average, 19.5 diglycolic anhydrides were modified onto the surface of human serum albumin (HSA). It was confirmed that A-HSA kept the stability and biocompatibility of native HSA. Moreover, it could inhibit Aβ42 fibrillogenesis and change the pathway of Zn(2+)-mediated Aβ42 aggregation, as demonstrated by extensive biophysical assays. In addition, upon incubation with A-HSA, the cytotoxicity presented by Zn(2+)-Aβ42 aggregates was significantly mitigated in living cells. The results showed that A-HSA had much stronger inhibitory effect on Zn(2+)-mediated Aβ42 fibrillogenesis and cytotoxicity than equimolar HSA. Isothermal titration calorimetry and stopped-flow fluorescence measurements were then performed to investigate the working mechanism of A-HSA. The studies showed that the A-HSA surface, with more negative charges, not only had stronger affinity for Zn(2+) but also might decrease the binding affinity of Aβ42 for Zn(2+). Moreover, hydrophobic binding and electrostatic repulsion could work simultaneously on the bound Aβ42 on the A-HSA surface. As a result, Aβ42 conformations could be stretched, which avoided the formation of toxic Zn(2+)-Aβ42 aggregates. The research thus revealed that A-HSA is a multifunctional agent capable of altering the pathway of Zn(2+)-mediated Aβ42 aggregation and greatly mitigating the amyloid cytotoxicity.

No abstract

Aggregation of amyloid β-proteins (Aβ) mediated by metal ions such as Zn has been suggested to be implicated in the progression of Alzheimer's disease (AD). Hence, development of bifunctional agents capable of inhibiting Aβ aggregation and modulating metal-Aβ species is an effective strategy for the treatment of AD. In this work, we modified iminodiacetic acid (IDA) onto human lysozyme (hLys) surface to create an inhibitor of Zn-mediated Aβ aggregation and cytotoxicity. The IDA-modified hLys (IDA-hLys) retained the stability and biocompatibility of native hLys. Extensive biophysical and biological analyses indicated that IDA-hLys significantly attenuated Zn-mediated Aβ aggregation and cytotoxicity due to its strong binding affinity for Zn, whereas native hLys showed little effect. Stopped-flow fluorescence spectroscopy showed that IDA-hLys could protect Aβ from Zn-induced aggregation and rapidly depolymerize Zn-Aβ aggregates. The research indicates that IDA-hLys is a bifunctional agent capable of inhibiting Aβ fibrillization and modulating Zn-mediated Aβ aggregation and cytotoxicity as a strong Zn chelator.

Metal-induced amyloid β-protein (Aβ) aggregation plays a key role in the pathogenesis of Alzheimer's disease. Although several agents have been recognized to block metal-associated Aβ aggregation, their therapeutic potential is marred due to the high-concentration metal ions in the amyloid plaques. To overcome this problem, we have herein developed iminodiacetic acid-modified human serum albumin (I-HSA) to fight against the aggregation. The multifunctional nature of I-HSA was extensively characterized in inhibiting the Aβ aggregation associated with Zn and Cu. The results revealed the following: (1) I-HSA significantly inhibited Aβ aggregation and alleviated its cytotoxicity. (2) I-HSA possessed a metal-chelate capacity as high as 31.2 mol/mol, and 25 μM I-HSA could effectively inhibit the influence of 250 μM Zn on Aβ aggregation. (3) Equimolar I-HSA remarkably attenuated the reactive oxygen species damage caused by the Aβ and Cu-Aβ species. (4) I-HSA could remodel metal-Aβ fibrils into unstructured aggregates with less neurotoxicity. The cytotoxicity of mature Cu-Aβ aggregates was mitigated from 64.8% to 25.4% under the functioning of I-HSA. In conclusion, I-HSA showed prominent advantages for the high metal-chelate capacity. To our knowledge, I-HSA is the first multifunctional macromolecule for inhibiting high-concentration metal-induced Aβ aggregation and remodeling mature metal-induced Aβ species.