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Bovine milk alpha(S2)-casein, an intrinsically disordered protein, readily forms amyloid fibrils in vitro and is implicated in the formation of amyloid fibril deposits in mammary tissue. Its two cysteine residues participate in the formation of either intra- or intermolecular disulphide bonds, generating monomer and dimer species. X-ray solution scattering measurements indicated that both forms of the protein adopt large, spherical oligomers at 20 degrees C. Upon incubation at 37 degrees C, the disulphide-linked dimer showed a significantly greater propensity to form amyloid fibrils than its monomeric counterpart. Thioflavin T fluorescence, circular dichroism and infrared spectra were consistent with one or both of the dimer isomers (in a parallel or antiparallel arrangement) being predisposed toward an ordered, amyloid-like structure. Limited proteolysis experiments indicated that the region from Ala(81) to Lys(113) is incorporated into the fibril core, implying that this region, which is predicted by several algorithms to be amyloidogenic, initiates fibril formation of alpha(S2)-casein. The partial conservation of the cysteine motif and the frequent occurrence of disulphide-linked dimers in mammalian milks despite the associated risk of mammary amyloidosis, suggest that the dimeric conformation of alpha(S2)-casein is a functional, yet amyloidogenic, structure. Refereed/Peer-reviewed

When not incorporated into the casein micelle, κ-casein, a major milk protein, rapidly forms amyloid fibrils at physiological pH and temperature. In this study, the effects of milk components (calcium, lactose, lipids, and heparan sulfate) and crowding agents on reduced and carboxymethylated (RCM) κ-casein fibril formation was investigated using far-UV circular dichroism spectroscopy, thioflavin T binding assays, and transmission electron microscopy. Longer-chain phosphatidylcholine lipids, which form the lining of milk ducts and milk fat globules, enhanced RCM κ-casein fibril formation irrespective of whether the lipids were in a monomeric or micellar state, whereas shorter-chain phospholipids and triglycerides had little effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid deposition in extracellular tissue, had little effect on the kinetics of RCM κ-casein fibril formation. Major nutritional components such as calcium and lactose also had no significant effect. Macromolecular crowding enhances protein-protein interactions, but in contrast to other fibril-forming species, the extent of RCM κ-casein fibril formation was reduced by the presence of a variety of crowding agents. These data are consistent with a mechanism of κ-casein fibril formation in which the rate-determining step is dissociation from the oligomer to give the highly amyloidogenic monomer. We conclude that the interaction of κ-casein with membrane-associated phospholipids along its secretory pathway may contribute to the development of amyloid deposits in mammary tissue. However, the formation of spherical oligomers such as casein micelles is favored over amyloid fibrils in the crowded environment of milk, within which the occurrence of amyloid fibrils is low.

Alzheimer’s disease (AD) is among the most important health-care problems in the world. The two pathological hallmarks of AD are extracellular neuritic amyloid plaques and intracellular neurofibrillary tangles. The aggregation of Aβ and β-sheet formation are considered to be the critical events which render these peptides neurotoxic. AD is affecting a large percentage of the elderly around the world. Many studies have been done on drugs to cure or at least slow Alzheimer’s disease. Most drugs produced for this disease aim at compensating for the performance of specific cell groups affected by the disease or restoring the function of these cells.This study examined the interaction of crocin, the main pigment of saffron, with the amyloid-β peptides 1 + 40 (Aβ 40) to determine the effects on peptide conformation and fibril formation using fluorescence spectroscopy, CD spectroscopy and electron microscopy. ThT data demonstrated the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of Aβ40. Incubation of pre-formed Aβ40 fibrils with crocin resulted in extensive lateral aggregation and precipitation of the fibrils. Consistent with this, electron microscopy data indicated that crocin decreased the number of fibrils formed and significantly reduced the average fibril length of Aβ40 as assessed by low levels of thioflavin T binding data. The mechanism by which, crocin prevented fibril formation was demonstrated by ANS binding assay and CD spectroscopy. In summary, crocin interacts with Aβ peptides and prevents amyloid formation. This means that it has the potential to be an important therapeutic drug against AD.

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, Aβ40, and the less common but more amyloidogenic form, Aβ42. 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 Aβ42 amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aβ42 amyloids. To further investigate the relationship between crocin and Aβ42 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 Aβ42. 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 Aβ42. 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 disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.