Investigation of metal-ion induced aggregation of human eye lens proteins by combination of X-ray spectroscopy, site-directed mutagenesis and quantum chemistry

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Worldwide average life expectancy has increased by 50 % since 1950 and surpassed 80 years in Europe. This remarkable global achievement of humankind demands a specific focus on age-related diseases to meet the growing challenges in the future. Age-related cataract is the leading cause of blindness on earth, especially in low- and middle-income countries. Presently, the only available treatment is surgery, which is the most common surgical procedure in Germany. Though effective, surgery has side effects and total annual cost in Germany will exceed one billion € in the next two decades. A lower cost alternative would be small molecule therapies delaying or even preventing cataract. Understanding the mechanism of cataract formation will facilitate biomedical research for such alternatives. Cataract results from aggregation of damaged lens proteins (crystallins), which leads to light-scattering high-molecular-weight complexes and lens opacity. Though the most important risk factor for cataract is age, several other factors, e.g. UV-radiation or diabetes, may accelerate cataract formation. Elevated risk for cataract is well documented in metal-working industries. Recently, essential transition metal ions, such as copper and zinc, have been identified as promoters of crystallin aggregation in vitro. Specifically, experiments in the group of Prof. L. Quintanar at the Cinvestav Institute, in collaboration with the research group of Prof. J. King at MIT, have discovered that very low (physiological) levels of Cu(II) and Zn(II) ions induce rapid aggregation of crystallin proteins resulting in light-scattering aggregates. The finding that essential metal ions can induce the aggregation of one of the most thermodynamically stable proteins in the human body is very surprising, and it reveals a novel and unexplored bio-inorganic facet of cataract disease. In this project, a combination of site-directed mutagenesis, synchrotron-based X-ray absorption and emission spectroscopy (XAS/XES) and quantum chemistry calculations is proposed to yield insight into the metal-induced aggregation of gamma-crystallin proteins. First, the nature of metal binding sites in gamma-crystallins will be characterized XAS/XES, supported by electron paramagnetic resonance and circular dichroism. Studying wild-type and mutated human gamma-crystallin proteins in solution containing zinc or copper will yield insight into redox changes and is expected to identify and characterize copper and zinc coordination sites. Second, experimentally-derived models for the metal-bound gamma-crystallin species will be used to build holistic theoretical representations employing a multi-scale (QM/MM) description to elucidate the mechanisms of metal-induced aggregation of human lens crystallins.