Natural dipeptides as mini-chaperones: molecular mechanism of inhibition of lens ßL-crystallin aggregation.

The effect of histidine-containing dipeptides-carnosine and N-acetylcarnosine-on preventing and treating of cataracts of various etiologic origins has been demonstrated in many studies in vivo, while the precise molecular mechanism of their action is actually obscure. Cataract has been recently attributed to conformational diseases due to the association of lens structure protein aggregation with cataract pathogenesis. In our study, effect of histidine-containing dipeptides-carnosine, N-acetylcarnosine, and anserine-on the UV induced ßL-crystallin aggregation was studied in vitro. It was first demonstrated that N-acetylcarnosine and anserine (10-40 mM) considerably suppressed UV induced aggregation of ßL-crystallin, while carnosine exerted no effect. Positive correlation between anti-aggregating activity of the compounds used and their hydrophobicity was obtained. It was revealed that N-acetylcarnosine and anserine inhibited the initial stages of the protein photochemical damage. A decrease in the size of protein aggregates was detected in the presence of N-acetylcarnosine and anserine. UV irradiation of ßL-crystallin resulted in a significant increase in the number of protein carbonyl groups, and the dipeptides studied did not affect this process. We suppose that N-acetylcarnosine and anserine inhibit ßL-crystallin aggregation via formation of a protein-dipeptide complex that prevents macromolecular conformational changes and ensuing protein aggregation.

Mechanism of aggregation of UV-irradiated ß(L)-crystallin.

Thermal denaturation and aggregation of UV-irradiated ß(L)-crystallin from eye lenses of steers have been studied. The data on size-exclusion chromatography and SDS-PAGE indicated that UV irradiation of ß(L)-crystallin at 10 °Ð¡ resulted in fragmentation of the protein molecule and formation of cross-linked aggregates. Fluorescence data showed that tryptophan fluorescence in the irradiated protein decreased exponentially with the UV dose. Decrease in tryptophan fluorescence is a result of photochemical destruction, but not of conformational changes of protein, because there is no red shift in the fluorescence maximum. The differential scanning calorimetry (DSC) profiles of the samples of UV-irradiated and wild type ß(L)-crystallin were registered. The area under curves, which is proportional to the amount of the native protein, decreased exponentially with increasing the irradiation dose. The shape of the DSC profiles for the samples of UV-irradiated ß(L)-crystallin was identical to that for wild type ß(L)-crystallin. The DSC data allowed estimating the portion of UV-denatured ß(L)-crystallin, which is not registered by DSC, and the portion of the combined fraction consisting of native and UV-damaged molecules retaining the native structure. A conclusion has been made that UV-induced denaturation of ß(L)-crystallin follows the one-hit model. The study of the kinetics of thermal aggregation of UV-irradiated ß(L)-crystallin at 37 °Ð¡ using dynamic light scattering showed that the initial stage of aggregation was that of formation of the start aggregates with the hydrodynamic radius of 20 nm. Further sticking of the start aggregates proceeded in the regime of reaction-limited cluster-cluster aggregation. Splitting of the aggregate population into two components occurred above a definite point in time.

Mechanism of thermal aggregation of yeast alcohol dehydrogenase I: role of intramolecular chaperone.

Kinetics of thermal aggregation of yeast alcohol dehydrogenase I (yADH) have been studied using dynamic light scattering at a fixed temperature (56 degrees C) and under the conditions where the temperature was elevated at a constant rate (1 K/min). The initial parts of the dependences of the hydrodynamic radius on time (or temperature) follow the exponential law. At rather high values of time splitting of the population of aggregates into two components occurs. It is assumed that such peculiarities of the kinetics of thermal aggregation of yADH are due to the presence of a sequence -YSGVCHTDLHAWHGDWPLPVK- in the polypeptide chain possessing chaperone-like activity. Thermodynamic parameters for thermal denaturation of yADH have been calculated from the differential scanning calorimetry data.

[Search for chaperon-like anticataract drugs, the antiaggregants of lens crystallins. Communication. 1. Chaperon-like activity of N-acetyl carnosine dipeptide: in vitro study on a model of ultraviolet-induced aggregation of betaL-crystallin].

Aggregation ofcrystallins, the lens proteins, is one of the basic stages of cataract formation. Among the protein aggregation models used to study the molecular mechanisms of the initial stages of lenticular opacity, UV-induced aggregation of betaL-crystallin is most close to the in vivo conditions. The carnosine derivative N-acetyl carnosine has been shown to be effective in inhibiting the UV-induced aggregation of betaL-crystallin. Examination of the accumulation kinetics of carbonyl groups in betaL-crystallin under UV irradiation has indicated that neither carnosine nor N-acetyl carnosine fails to affect this parameter--an indicator of oxidative protein damage. By taking into account also the fact that N-acetyl carnosine is not an antioxidant, it can be believed that the molecular mechanism of action of this compound on UV-induced aggregation of betaL is unassociated with its antioxidative properties. The authors hypothesize that the molecular chaperon-like properties similar to those of alpha-crystallin underlie the mechanism of action of the acetyl derivative carnosine. The prospects for searching anticataract agents of a new chaperon-like class are discussed.

[Like anticataract agents, the antiaggregants of lens crystallin. Communication 2. Study of the impact of chaperon-like (protective) activity of short-chain peptides on the rate of UV-induced aggregation of betaL-crystallins by eximer laser].

UV-induced aggregation of betaL-crystallin, one of the major lens proteins, was studied under its pulse radiation with XeCl laser at a wavelength of 308 nm. Unlike the in vitro tested dipeptides L-carnosine, N-acetyl carnosine, D-panthetine, and particularly their combination, the so-called new chaperon was demonstrated to slow down the rate of photoaggregatin of beta-crystallin. The new chaperon, a mixture of D-pathethine and N-acetyl carnosine was ascertained to protect a mixture of betaL- and alpha-crystallins from UV-induced aggregation to a greater extent than D-pathethine or N-acetyl carnosine used alone. An effective drug based on the new chaperon may be designed for the prevention of cataract in sight.

Nonreciprocal XeCl laser-induced aggregation of beta-crystallins in water solution.

The aggregation of a beta-crystallin water solution exposed to XeCl laser radiation demonstrates the dependence of scattering-exposure curve (scattering versus exposure) on laser intensity. The main features of this dependence can be understood by the relaxation of a partly denaturated state of a protein within some finite relaxation time. These photoactivated states originate from the absorption of UV photons. Two partly denaturated (photoactivated) monomers, as well as other aggregates, can aggregate, giving rise to sharply increasing probe light scattering after some lag time of irradiation.