Effects Of Zn²⁺ And Brazilin On The Aggregation Of Amyloid Proteins

Alzheimer’s disease(AD)and type 2 diabetes mellitus(T2DM)are two common types of conformational diseases.The misfolding and aggregation of amyloid β-protein(Aβ)and human islet amyloid polypeptide(hIAPP)are considered to be the main causes of AD and T2 DM,respectively.At present,inhibitors against the aggregation of amyloid proteins often lack effectiveness due to their single function.Hence a comprehensive understanding of the aggregation mechanisms of amyloid proteins in vivo will benefit the development of universal multi-functional inhibitors.In vitro studies have demonstrated that Zn2+ has great impact on the aggregation of amyloid proteins.However,in the brain Zn2+ actually interact with amyloid proteins through the transiently released Zn2+ “pulses” during neurotransmission.The lifespans of Zn2+ “pulses” are very short,thus it is difficult to explore the details of the interactions between Zn2+ and amyloid proteins within such a short time scale by using ordinary analytical methods.In the meanwhile,the functional mechanisms of some small molecular inhibitors on the Zn2+-induced aggregation of amyloid proteins are still poorly understood.To counter the problems above,firstly a stopped-flow apparatus with a dead time of 1 ms was utilized to study the interactions between Zn2+ and Aβ within the time scale of Zn2+ “pulses” during neurotransmission.It was found that Zn2+ bound with Aβ42 within 1 ms,caused immediate conformational change around Tyr10,and promoted Aβ42 aggregation by enhancing hydrophobic interactions.Three kinetic phases were observed in the promoted aggregation,and the rate constant of the fast phase was increased by almost 200 folds upon the addition of 100 μmol/L Zn2+.Moreover,the promoting effect of Zn2+ on Aβ42 fast aggregation was closely related to pH value.Compared with the pH range in which Aβ42 fast aggregation occurred in the absence of Zn2+(pH5.2-6.2),it broadened to pH5.2-7.8 upon the addition of Zn2+,indicating that the existence of Zn2+ enabled the fast aggregation of Aβ42 to happen in circumstances with wider pH ranges.Based on the studies above,various methods were taken to measure the ability of brazilin to chelate Zn2+,as well as to inhibit the Zn2+-induced Aβ42 aggregation.Brazilin was found to has moderate affinity to chelate Zn2+(KD=46±6.8 μmol/L),and could compete with Zn2+ for Aβ42.Consequently,brazilin potently inhibited the Zn2+-induced fast aggregation of Aβ42,and attenuated the resulted cytotoxicity.Furthermore,it was demonstrated that brazilin postponed the conformational transition of h IAPP from α-helix to β-sheet,thus inhibited the aggregation of h IAPP.Besides,brazilin could also disaggregate the preformed fibrils,and attenuate the cytotoxicity caused by hIAPP aggregation.In the end,the influences of brazilin and Zn2+ on the seeding and cross-seeding of Aβ and hIAPP were investigated.It was found that Zn2+ inhibited the seeding and crossseeding ability of Aβ40,while promoted that of hIAPP.In the meanwhile,brazilin effectively inhibited the seeding effect of Aβ40.This study provided new insights into the influence of Zn2+ on the aggregation of amyloid proteins,and demonstrated that brazilin is a universal multi-functional inhibitor.The findings would benefit in a comprehensive understanding of AD and T2 DM pathologies,as well as the development of effective inhibitors against the aggregation of amyloid proteins.