Abstract:Aβ is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer's disease. There are two common isoforms of Aβ: the more common form, Aβ 40 , and the less common but more amyloidogenic form, Aβ 42 . Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aβ 42 amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aβ 42 amyloids. To further investigate the relationship between crocin and Aβ 42 structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aβ 42 . It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aβ. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aβ 42 . CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and β-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aβ amyloid formation but also by the Unauthenticated Download Date | 5/12/18 7:38 AM CELLULAR & MOLECULAR BIOLOGY LETTERS 329 disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.

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Amyloid fibrils arise from the slow aggregation of intermediately folded protein states. In this study the kinetics of the protein fibril formation of alpha-lactalbumin and its prevention by alphaS-casein in the presence and absence of the crowding agent, dextran (68 kDa), have been compared using a thioflavin T binding assay. It was found that alphaS-casein, a molecular chaperone found in bovine milk, is a potent in vitro inhibitor of alpha-lactalbumin fibrillization. The effect of alphaS-casein in preventing fibril formation was significant, although less than it is in the absence of the crowding agent, dextran. The interaction between the chaperone and the alpha-lactalbumin and structural change in the target protein are also shown using intrinsic fluorescence intensity, an ANS binding assay, CD spectroscopy and size-exclusion HPLC. In summary, alpha-casein interacts with alpha-lactalbumin and prevents amyloid formation but not as well as it does when the crowding agent, dextran, not present.

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Abstract:The current study investigates the inhibitory effect of crocin(s), also known as saffron apocarotenoids, on protein glycation and aggregation in diabetic rats, and α-crystallin glycation. Thus, crocin(s) were administered by intraperitoneal injection to normal and streptozotocin-induced diabetic rats. The cataract progression was recorded regularly every two weeks and was classified into four stages. After eight weeks, the animals were sacrificed and the parameters involved in the cataract formation were measured in the animal lenses. Some parameters were also determined in the serum and blood of the rats. In addition, the effect of crocin(s) on the structure and chaperone activity of α-crystallin in the presence of glucose was studied by different methods. Crocin(s) lowered serum glucose levels of diabetic rats and effectively maintained plasma total antioxidants, glutathione levels and catalase activity in the lens of the animals. In the in vitro study, crocin(s) inhibited α-crystallin glycation and aggregation. Advanced glycation end products fluorescence, hydrophobicity and protein cross-links were also decreased in the presence of crocin(s). In addition, the decreased chaperone activity of α-crystallin in the presence of glucose changed and became close to the native value by the addition of crocin(s) in the medium. Crocin(s) thus showed a powerful inhibitory effect on α-crystallin glycation and preserved the structure-function of this protein. Crocin(s) also showed the beneficial effects on prevention of diabetic cataract.

Tissue deposition of normally soluble proteins, or their fragments, as insoluble amyloid fibrils causes both acquired and hereditary systemic amyloidoses, which is usually fatal. Amyloid is associated with serious diseases such as Alzheimer's disease, type 2 diabetes, Parkinson's Disease, Huntington's Disease, cancer and the transmissible spongiform encephalopathies. Information concerning the structure and mechanism of formation of fibrils in these diseases is critical for understanding the process of pathology of the amyloidoses and to the development of more effective therapeutic agents that target the underlying disease mechanisms. Structural models have been made using information from a wide variety of techniques, including electron microscopy, X-ray diffraction, solid state NMR, and Congo red and CD spectroscopy. Although each type of amyloidosis is characterised by a specific amyloid fibril protein, the deposits share pathognomonic histochemical properties and the structural morphology of all amyloid fibrils is very similar. In fact, the structural similarity that defines amyloid fibres exists principally at the level of β-sheet folding of the polypeptides within the protofilament, while the different types vary in the supramolecular assembly of their protofilaments.