Hamilton Paul D, Bozeman Stephanie L, Andley Usha P
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, 63110, USA.
Biochem Biophys Rep. 2020 Feb 29;22:100748. doi: 10.1016/j.bbrep.2020.100748. eCollection 2020 Jul.
Creatine kinase (CK) is an energy storage enzyme that plays an important role in energy metabolism. CK/phosphocreatine functions as an energy buffer and links ATP production sites with ATP utilization sites. Several key mutations in the αA-crystallin () and αB-crystallin () genes have been linked with autosomal-dominant, hereditary human cataracts. The cryaa-R49C mutation was identified in a four-generation Caucasian family. We previously identified an increase in the quantity of CK complexed with α-crystallin in the lenses of knock-in mice expressing the cryaa-R49C mutation using proteomic analyses. Increased levels of CK in postnatal cataractous lenses may indicate increased ATP requirements during early cataract development. To gain a further understanding of the relationship between CK and α-crystallin, we investigated whether α-crystallin interacts with and forms complexes with CK, . Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 α-crystallin subunits, with a K of 3.3 × 10 M, and that the interaction between α-crystallin and CK was endothermic, thermodynamically favorable, and entropy-driven. High-salt concentrations did not affect the interaction between CK and α-crystallin, suggesting that the interaction between CK and α-crystallin is primarily hydrophobic. Gel permeation chromatography (GPC) detected water-soluble α-crystallin and CK complexes, as determined by increased light scattering after complex formation. In addition, CK and α-crystallin formed partially-water-insoluble, high-molecular-mass complexes. Enzyme-linked immunosorbent assay (ELISA)-based enzymatic activity analyses of lens homogenates showed a 17-fold increase in CK activity in the postnatal lenses of cryaa-R49C knock-in mice. These studies indicate that the interaction between α-crystallin and CK is functionally important and that increased CK levels may be necessary to meet the increased ATP demands of ATP-dependent functions in cataractous lenses.
肌酸激酶（CK）是一种能量储存酶，在能量代谢中起重要作用。CK/磷酸肌酸作为一种能量缓冲物质，将ATP产生位点与ATP利用位点联系起来。αA-晶状体蛋白（）和αB-晶状体蛋白（）基因中的几个关键突变与常染色体显性遗传性人类白内障有关。在一个四代白人家庭中发现了cryaa-R49C突变。我们之前通过蛋白质组学分析发现在表达cryaa-R49C突变的敲入小鼠晶状体中，与α-晶状体蛋白复合的CK数量增加。出生后白内障晶状体中CK水平升高可能表明在白内障早期发展过程中ATP需求增加。为了进一步了解CK与α-晶状体蛋白之间的关系，我们研究了α-晶状体蛋白是否与CK相互作用并形成复合物。等温滴定量热法（ITC）表明，每个CK二聚体与28个α-晶状体蛋白亚基结合，解离常数K为3.3×10⁻⁹M，并且α-晶状体蛋白与CK之间的相互作用是吸热的，在热力学上是有利的，且由熵驱动。高盐浓度不影响CK与α-晶状体蛋白之间的相互作用，这表明CK与α-晶状体蛋白之间的相互作用主要是疏水的。凝胶渗透色谱法（GPC）检测到了水溶性的α-晶状体蛋白和CK复合物，复合物形成后光散射增加即证明了这一点。此外，CK和α-晶状体蛋白形成了部分水不溶性的高分子量复合物。基于酶联免疫吸附测定（ELISA）对晶状体匀浆进行的酶活性分析表明，cryaa-R49C敲入小鼠出生后晶状体中的CK活性增加了17倍。这些研究表明，α-晶状体蛋白与CK之间的相互作用在功能上很重要，并且升高的CK水平可能是满足白内障晶状体中ATP依赖性功能增加的ATP需求所必需的。
