Screening Inhibitors Of Amyloid Fibril Formation By κ-casein And Studying Of Their Mechanisms

It is well known that κ-casein readily forms amyloid fibrils under physiological conditions, and the deposition of amyloid fibril found within mammary tissue is recognized to be a major contributing factor in a group of pathologic states known as amyloidosis of the breast. Herein, understanding the aggregation process and knowing how to prevent amyloid fibril formation have been considered as a promising approach to advance the clinical treatment of these diseases. RCMκ-CN serves as an excellent model to study generic aspects of fibril formation and its inhibition, which is prepared by reduction of the κ-casein disulfide linkages and subsequent carboxymethylation. We have investigated the molecular interaction of L-arginine(ARG) and ginsenoside(GS) with κ-casein, and their effects on amyloid fibril formation of the protein by several spectroscopic methods. Based on experiment data, the mechanism by which ARG and GS influenced fibril formation are also explored herein.(1) The interaction of ARG with κ-casein, and its effect on amyloid fibril formation of the protein, have been investigated in vitro by resonance light scattering(RLS), fluorescence, UV–Vis absorption spectroscopy and transmission electron microscopy(TEM) under simulated physiological conditions. The results indicated that ARG inhibited fibril formation by reduced and carboxymethylated κ-casein(RCMκ-CN), and there was interaction between ARG and RCMκ-CN, proved by the observation of enhancement in RLS intensity attributed to the formation of RCMκ-CN–ARG complex. It was also demonstrated that ARG strongly quenched the intrinsic fluorescence of RCMκ-CN through a static quenching mechanism. The corresponding thermodynamic parameters(ΔH, ΔS and ΔG) were tested to show that the binding process was spontaneous and mainly enthalpy driven with an unfavorable entropy, and both hydrogen bond and van der Waals forces played a key role in the binding of ARG and RCMκ-CN. The determined value of the distance r between ARG and RCMκ-CN Trp97 residue evaluated by fluorescence resonance energy transfer(FRET) was 2.94 nm. Furthermore, the conformational investigation from synchronous fluorescence showed that the RCMκ-CN Trp97 residue was placed in a less polar environment and more difficultly exposed to the solvent after addition of ARG, which was conjectured to be the reason that ARG inhibited structure changes of RCMκ-CN from native state to β shift and fibril formation by κ-casein.(2) We have investigated the ability of ginsenoside Rb1, Rg1, Rc and Re to influence fibril formation by RCMκ-CN, with the methods of Thioflavin T fluorescence assay, transmission electron microscopy(TEM) and intrinsic fluorescence spectroscopy. The results showed that Rb1 and Rg1 inhibited obviously RCMκ-CN fibrillation in both the initial rate and final level of Th T fluorescence. On the contrary, Re has a few effect on promoting fibril formation by RCMκ-CN, proved by thick and larger fibrils observed frequently in TEM, while Rc were not found to have effect on fibril formation by RCMκ-CN. Intrinsic fluorescence spectroscopy showed that the λmax of RCMκ-CN decreased from 336 to 331 nm after incubated lonely in 24 h, indicating that environment of Trp97 altered during the incubation of RCMκ-casein. But the λmax of RCMκ-CN remained at 336 nm when incubated in presence of Rg1, which demonstrated that Rg1 prevent RCMκ-CN fibril formation by stabilising RCMκ-CN in its native like state. Additional chemical structure difference of ginsenosides and the effects on fibril formation are also implicated.(3) The interaction of GS including ginsenoside Rg1, Rb1 and Re with RCMκ-CN have been investigated in vitro by fluorescence and ultraviolet spectra. The enhancement in RLS intensity was attributed to the formation of new complex between GS and RCMκ-CN, and the corresponding thermodynamic parameters(ΔH, ΔS and ΔG) were assayed. The steady-state ultraviolet-visible absorption spectra had also been tested to observe if the ground-state complex formed, and it showed the same result as RLS spectra. The binding constants and the number of binding sites between GS and RCMκ-CN at different temperatures had been evaluated from relevant fluorescence data. According to F?rster non-radiation energy transfer theory, the binding distance between RCMκ-CN and GS was calculated. The fluorescence lifetime of RCMκ-CN was longer in the presence of GS than in absence of GS, which was evident that the hydrophobic interaction play a major role in the binding of GS to RCMκ-CN. From the results of synchronous fluorescence, it could be deduced that the polarity around RCMκ-CN Trp97 residue decreased and the hydrophobicity increased after addition of Rg1 or Rb1. Based on all the above results, it is explained that Rg1 and Rb1 inhibited amyloid fibril formation by κ-casein because the molecular spatial conformation and physical property of κ-casein changed causing by the complex formation between GS and κ-casein.