Palomino-Vizcaino Giovanni, Schuth Nils, Domínguez-Calva José A, Rodríguez-Meza Oscar, Martínez-Jurado Eduardo, Serebryany Eugene, King Jonathan A, Kroll Thomas, Costas Miguel, Quintanar Liliana
Department of Chemistry, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City 07360, Mexico.
Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510, Mexico.
J Am Chem Soc. 2023 Mar 29;145(12):6781-6797. doi: 10.1021/jacs.2c13397. Epub 2023 Mar 14.
Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu ions with γD-, γC-, and γS-crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of β-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu binding sites in each protein. Spin quantitation demonstrated the reduction of γ-crystallin-bound Cu ions to Cu under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins' Cu reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.
白内障是由人眼晶状体蛋白的高分子量聚集体引起的，这些聚集体散射光线，导致晶状体混浊。金属离子已成为白内障疾病病因中的重要潜在因素，因为人晶状体γ-晶状体蛋白易受金属诱导的聚集作用影响。在此，已评估了铜离子与γD-、γC-和γS-晶状体蛋白（晶状体中三种最丰富的γ-晶状体蛋白）之间的相互作用。铜离子在所有这三种蛋白质中均诱导了非淀粉样聚集。溶液比浊法、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、圆二色性和差示扫描量热法表明，铜诱导聚集的机制涉及：（i）N端结构域中β-折叠结构的丧失；（ii）热稳定性和动力学稳定性降低；（iii）金属桥连物种的形成；以及（iv）二硫键桥连二聚体的形成。等温滴定量热法（ITC）揭示了γ-晶状体蛋白中不同的铜结合亲和力。电子顺磁共振（EPR）揭示了每种蛋白质中有两个不同的铜结合位点。自旋定量表明，在有氧条件下，γ-晶状体蛋白结合的铜离子还原为铜，而X射线吸收光谱（XAS）证实了γ-晶状体蛋白中存在线性或三角铜结合位点。我们的EPR和XAS研究表明，γ-晶状体蛋白的铜还原酶活性产生一种基于蛋白质的自由基，在人γD-晶状体蛋白中可能是基于酪氨酸的物种。人晶状体γ-晶状体蛋白中不同的氧化还原活性铜位点进行的这种独特的自由基化学作用可能有助于铜诱导聚集的机制。在衰老的人晶状体背景下，γ-晶状体蛋白不仅可以作为结构蛋白，还可以作为金属和氧化还原稳态的关键参与者。
