Photosensitized structural modifications of the lens protein [alpha]-crystallin: Do all modifications impair chaperone-like activity?[para]

Photochemistry and Photobiology, May 2003 by Sgarbossa, Antonella, Youssef, Tareq, Lenci, Francesco

ABSTRACT

Among chaperone-like functioning proteins, the lens [alpha]-crystallins are of particular interest because they are not renewed, and even minor alterations can hurt their function of maintaining the proper refractive index and avoiding cataract formation in the lens. Several reports have suggested the occurrence of remarkable structural modifications in lens proteins in the presence of endogenous and exogenous sensitizers upon exposure to light. In particular, it has been shown in vitro that hypericin, the active ingredient of Hypericum, can bind to and, in the presence of light, cause the photopolymerization of [alpha]-crystallin. On the basis of these results it has also been suggested that a subsequent significant impairment of the protein function can occur. Using absorption and emission spectroscopic techniques, as well as circular dichroism, we have studied the structural modifications of [alpha]-crystallin resulting from its interaction with hypericin after irradiation with visible light. To investigate the chaperone-like function of [alpha]-crystallin, the heat-induced aggregation kinetics of another lens protein, [beta]^sub Low^-crystallin, was monitored by measuring the apparent absorption due to scattering at 360 nm as a function of time, and no apparent damage to its functional role was observed. Spectroscopic results, on the contrary, show a prominent reduction in both tryptophan and hypericin fluorescence emission intensity after light irradiation, suggesting an alteration in the tryptophan microenvironment and a high degree of packing of the chromophore due to photoinduced modification of the molecular framework. Control experiments on [alpha]-crystallin structurally modified by light in the presence of hypericin indicated that the protein still retains its ability to chaperone both lens crystallins and insulin.

Abbreviations: CD, circular dichroism; DTT, dithiothreitol; EtOH, ethanol; PBS, phosphate-buffered solution; ROS, reactive oxygen species; Trp, tryptophan.

INTRODUCTION

The lens [alpha]-crystallin, the major structural protein of the vertebrate eye lens, constitutes up to 40% of the total lens proteins and accomplishes the critical function of maintaining transparency and proper refractive index in the lens (1-3). [alpha]-Crystallin is an aggregate of about 800 kDa, in which two evolutionary related subunits, [alpha]A and [alpha]B, with molecular masses of approximately 20 kDa each, form the building blocks of the aggregate (1,3-5). A great deal of attention has been devoted to the study of the stress-induced structural modifications of this protein and the possible functional consequences for its role in the eye lens. Structural alterations after exposure to UV-radiation, or to visible light in the presence of endogenous or exogenous photosensitizers, for example, have been suggested as seriously affecting the protein functional role (1,6-10). Photoinduced oxidative damages can occur directly upon absorption of UV radiation by aromatic residues or indirectly through the generation of reactive oxygen species (ROS) upon absorption of visible light by photosensitizers (1,5,6,11,12). Because the functional properties of the lens are largely dependent on solubility and stability of the crystallin proteins, which it contains at high concentrations (of the order of 500 mg/mL) and because of the lack of protein turnover, even minor local disturbances can bring about large-scale alterations of the molecular architecture and contribute to the growth of light-scattering aggregates. [alpha]-Crystallin plays its key role of preserving the lens functional integrity thanks to its chaperone-like activity: it specifically recognizes and binds to aggregation-prone nonnative structures occurring early in the denaturation pathway of proteins and hinders the degradation process itself (13-19). In vitro studies revealed that [alpha]-crystallin is capable of preventing nonspecific aggregation of the other lens proteins, [beta]- and [gamma]-crystallins (13,19,20), and even of different noncrystalline proteins and enzymes (e.g. insulin, [alpha]-lactalbumin, alcohol dehydrogenase and citrate synthase) (21,22) caused by thermal or other stresses.

Because of the previously mentioned lack of protein turnover in lens proteins and minimal repair mechanisms, the integral function of [alpha]-crystallin as a chaperone in vivo is of crucial importance in the prevention of cataract (1,3,5,13).

Among naturally occurring pigments, hypericin, which has for several years been used largely as a mild antidepressant (23-25), could also be a photosensitizer that is particularly effective in the treatment of serious ocular infections (it absorbs both in the UV range and in the visible range up to 590 nm) through singlet oxygen production. On the other hand, the eyes are continuously exposed to light, and the use of hypericin in either role out of medical control might also constitute a source of damage for the lens and the retina.