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

结合 X 射线光谱、定点突变和量子化学研究金属离子诱导的人眼晶状体蛋白聚集

• 批准号: 438291468
• 负责人: Dr. Nils Schuth
• 金额: --
• 依托单位: Departamento de Química
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/438291468?language=en
• 关键词: Investigation; metal; ion; induced; aggregation

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.

自1950年以来，全球平均预期寿命增加了50%，欧洲超过了80岁。人类这一了不起的全球成就要求特别重视与年龄有关的疾病，以应对未来日益增长的挑战。与年龄相关的白内障是地球上失明的主要原因，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。虽然有效，但手术有副作用，未来二十年德国每年的总成本将超过10亿欧元。一种成本较低的替代方案是小分子疗法，可以延缓甚至预防白内障。了解白内障形成的机制将有助于这些替代品的生物医学研究。白内障是由受损的透镜蛋白（晶状体蛋白）聚集引起的，其导致光散射高分子量复合物和透镜不透明。虽然白内障最重要的危险因素是年龄，但其他一些因素，例如紫外线辐射或糖尿病，可能会加速白内障的形成。在金属加工行业中，白内障的风险增加是有据可查的。最近，必需的过渡金属离子，如铜和锌，已被确定为晶体蛋白聚集在体外的促进剂。具体地说，L. Cininvstav研究所的Quintanar与麻省理工学院的J. King教授的研究小组合作，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中最稳定的蛋白质之一的聚集的发现是非常令人惊讶的，并且它揭示了白内障疾病的一个新的和未探索的生物无机方面。在这个项目中，结合定点诱变，同步加速器为基础的X射线吸收和发射光谱（XAS/XES）和量子化学计算，提出了深入了解金属诱导的γ-晶体蛋白的聚集蛋白。首先，γ-晶体蛋白中金属结合位点的性质将被表征为XAS/XES，由电子顺磁共振和圆二色性支持。研究野生型和突变的人γ-晶状体蛋白在含有锌或铜的溶液中的蛋白质将产生对氧化还原变化的洞察，并有望识别和表征铜和锌的配位位点。第二，实验衍生的金属结合的γ-晶体蛋白物种的模型将被用来建立整体的理论表示采用多尺度（QM/MM）的描述，以阐明金属诱导的人类透镜晶体蛋白聚集的机制。