Trehalose Inhibits the Heat-Induced Formation of the Amyloid-Like Structure of Soluble Proteins Isolated from Human Cataract Lens.

The age-dependent loss of solubility and aggregation of crystallins constitute the pathological hallmarks of cataract. Several biochemical and biophysical factors are responsible for the reduction of crystallins' solubility and formation of irreversible protein aggregates, which display amyloid-like characteristics. The present study reports the heat-induced aggregation of soluble proteins isolated from human cataract lenses and the formation of amyloid-like structures. Exposure of protein at 55 °C for 4 h resulted in extensive (≈ 60%) protein aggregation. The heat-induced protein aggregates displayed substantial (≈ 20 nm) redshift in the wavelength of maximum absorption (λmax) of Congo red (CR) and increase in Thioflavin T (ThT) fluorescence emission intensity, indicating the presence of amyloid-like structures in the heat-induced protein aggregates. Subsequently, the addition of trehalose resulted in substantial inhibition of heat-induced aggregation and the formation of amyloid-like structure. The ability of trehalose to inhibit the heat-induced aggregation was found to be linearly dependent upon its concentration used. The optimum effect was observed in the presence of 30-40% (w/v) trehalose where the aggregated was found to be reduced from 60 to 30%. The present study demonstrated the ability to trehalose to inhibit the protein aggregation and interfere with the formation of amyloid-like structures.

Predicted aggregation-prone region (APR) in ßB1-crystallin forms the amyloid-like structure and induces aggregation of soluble proteins isolated from human cataractous eye lens.

The aggregation of ß-crystallins in the human eye lens constitutes a critical step during the development of cataract. We anticipated that the presence of Aggregation-Prone Regions (APRs) in their primary structure, which might be responsible for conformational change required for the self-assembly. To examine the presence of APRs, we systematically analyzed the primary structures of ß-crystallins. Out of seven subtypes, the ßB1-crystallin found to possess the highest aggregation score with 9 APRs in its primary structure. To confirm the amyloidogenic nature of these newly identified APRs, we further studied the aggregation behavior of one of the APRs spanning from 174 to 180 residues (174LWVYGFS180) of ßB1-crystallin, which is referred as ßB1(174-180). Under in vitro conditions, the synthetic analogue of ßB1(174-180) peptide formed visible aggregates and displayed high Congo red (CR) bathochromic shift, Thioflavin T (ThT) binding and fibrilar morphology under transmission electron microscopy, which are the typical characteristics of amyloids. Further, the aggregated ßB1(174-180) was found to induce aggregation of the soluble fraction of proteins isolated from the human cataractous lens. This observation suggests that the presence of APRs in ßB1-crystallin might be serving as one of the intrinsic supplementary factors responsible for constitutive aggregation behavior of ßB1-crystallin and development of cataract.

Effect of Green Tea Polyphenol Epigallocatechin-3-gallate on the Aggregation of αA(66-80) Peptide, a Major Fragment of αA-crystallin Involved in Cataract Development.

PURPOSE: Crystallin is a major protein present in eye lens. Peptide fragment αA(66-80) derived from αA-crystallin possesses high aggregation propensity and forms amyloid-like structures. αA(66-80) aggregates are known to interact with soluble crystallins and destabilize native structures that subsequently undergo aggregation. Crystallin aggregation in eye lens leads to reduction in lens opacity, the condition generally referred to as a cataract. Thus, αA(66-80) aggregation appears to be an important event during cataract development, and therefore, inhibition of αA(66-80) aggregation may be an attractive strategy to intervene in cataract development. MATERIALS AND METHODS: αA(66-80) peptide derived from αA-crystallin possesses high aggregation potential and has a crucial role in cataract development. In order to inhibit the aggregation of αA(66-80) peptide, epigallocatechin-3-gallate (EGCG), a major active constituent of green tea, was employed. The inhibitory effect was assessed by Congo Red (CR) spectral shift assay, Thioflavin-T binding assay, transmission electron microscopy and fluorescence microscopy. RESULTS: The inhibitory potential of EGCG toward αA-crystallin was clearly observed as in the presence of EGCG, the αA(66-80) aggregation was considerably inhibited and the pre-formed fibrillary aggregates of αA(66-80) were found to be disassembled. CONCLUSION: In the present study, we are able to successfully demonstrate that EGCG efficiently blocks the aggregation of αA(66-80) peptide in a concentration-dependent manner. Furthermore, it is also evident that EGCG is able to disaggregate pre-formed αA(66-80) aggregates. The study suggests that EGCG can be a potential molecule that can prevent the initiation of cataract as well as be helpful in the disease reversal.