The aggregation of ␣-synuclein has been implicated as a critical step in the development of Parkinson's disease. Parkinson's disease is a progressive neurodegenerative disorder caused by the loss of dopaminergic neurons from the substantia nigra; currently, no cure exists. Baicalein is a flavonoid with antioxidant properties; upon oxidation, it forms several products including quinones. We show here that low micromolar concentrations of baicalein, and especially its oxidized forms, inhibit the formation of ␣-synuclein fibrils. In addition, existing fibrils of ␣-synuclein are disaggregated by baicalein. The product of the inhibition reaction is predominantly a soluble oligomer of ␣-synuclein, in which the protein molecules have been covalently modified by baicalein quinone to form a Schiff base with a lysine side chain in ␣-synuclein. The binding of baicalein was abolished by conversion of the Tyr residues into Phe, demonstrating that Tyr is involved in the interaction of ␣-synuclein with baicalein.
The aggregation of the presynaptic protein alpha-synuclein is associated with Parkinson's disease (PD). The details of the mechanism of aggregation, as well as the cytotoxic species, are currently not well understood. alpha-Synuclein has four tyrosine and no tryptophan residues. We introduced a tyrosine to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. Y39W alpha-synuclein had similar fibrillation kinetics (2-fold slower), pH-induced conformational changes, and fibril morphology to wild-type alpha-synuclein. In addition to intrinsic Trp fluorescence, acrylamide quenching, fluorescence anisotropy, ANS binding, dynamic light scattering, and FTIR were employed to monitor the kinetics of aggregation. These biophysical probes revealed the significant population of two classes of oligomeric intermediates, one formed during the lag period of fibrillation and the other present at the completion of fibrillation. As expected for a natively unfolded protein, Trp 39 was highly solvent-exposed in the monomer and is solvent-exposed in the two oligomeric intermediates; however, it is partially, but not fully, buried in the fibrils. These observations demonstrate the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.
Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternate visual cycle for regenerating opsins in daylight. Here, we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternate pathway. DES1 is expressed in retinal Müller cells where it co-immunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in rpe65 −/− mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA-interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 possessed very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity.
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