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

自1950年以来，全球平均预期寿命增加了50%，在欧洲超过了80岁。人类取得的这一显著的全球成就要求我们特别关注与年龄有关的疾病，以应对未来日益增长的挑战。年龄相关性白内障是全球致盲的主要原因，尤其是在低收入和中等收入国家。目前，唯一可用的治疗方法是手术，这是德国最常见的外科手术。手术虽然有效，但也有副作用，未来20年，德国每年的总费用将超过10亿欧元。一种低成本的替代方法是小分子疗法，可以延缓甚至预防白内障。了解白内障形成的机制将促进此类替代品的生物医学研究。白内障是由于晶状体受损蛋白（晶体蛋白）聚集导致光散射的高分子量复合物和晶状体混浊。虽然白内障最重要的危险因素是年龄，但其他一些因素，如紫外线辐射或糖尿病，也可能加速白内障的形成。在金属加工行业中，白内障的风险增加是有充分证据的。近年来，重要的过渡金属离子，如铜和锌，已被确定为晶体蛋白体外聚集的促进剂。具体来说，Cinvestav研究所的L. Quintanar教授小组与麻省理工学院J. King教授的研究小组合作进行的实验发现，非常低（生理）水平的Cu（II）和Zn（II）离子诱导晶体蛋白快速聚集，导致光散射聚集体。必需金属离子可以诱导人体内热力学最稳定的蛋白质之一的聚集，这一发现非常令人惊讶，它揭示了白内障疾病的一个新的和未被探索的生物无机方面。在这个项目中，提出了位点定向诱变、基于同步加速器的x射线吸收和发射光谱（XAS/XES）和量子化学计算的结合，以深入了解金属诱导的γ -结晶蛋白聚集。首先，在电子顺磁共振和圆二色性的支持下，用XAS/XES表征了γ -晶体蛋白中金属结合位点的性质。在含锌或铜的溶液中研究野生型和突变的人类γ -晶体蛋白将有助于了解氧化还原变化，并有望识别和表征铜和锌的配位位点。其次，实验衍生的金属结合γ -晶体蛋白物种模型将用于建立采用多尺度（QM/MM）描述的整体理论表示，以阐明金属诱导的人类晶状体晶体蛋白聚集的机制。

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.