Creatine kinase/α-crystallin interaction functions in cataract development
| Autor: | Stephanie L. Bozeman, Usha P. Andley, Paul D. Hamilton |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
CKM creatine kinase M PBS phosphate-buffered saline Biophysics medicine.disease_cause Biochemistry Cataract RALS right angle light scattering Mouse model RI refractive index Phosphocreatine lcsh:Biochemistry Gel permeation chromatography 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Crystallin medicine lcsh:QD415-436 Creatine kinase lcsh:QH301-705.5 chemistry.chemical_classification Mutation biology ITC isothermal titration calorimetry Chemistry Complex formation Isothermal titration calorimetry ELISA enzyme-linked immunosorbent assay GPC gel permeation chromatography WT wild-type eye diseases In vitro CKB creatine kinase B α-Crystallin 030104 developmental biology Enzyme lcsh:Biology (General) cryaa-R49C αA-crystallin R49C mutant 030220 oncology & carcinogenesis biology.protein sense organs CK creatine kinase Research Article |
| Zdroj: | Biochemistry and Biophysics Reports Biochemistry and Biophysics Reports, Vol 22, Iss, Pp-(2020) |
| ISSN: | 2405-5808 |
| DOI: | 10.1016/j.bbrep.2020.100748 |
| Popis: | 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 (cryaa) and αB-crystallin (cryab) 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, in vitro. Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 α-crystallin subunits, with a Kd of 3.3 × 10−7 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. Highlights • Cataract model α-crystallin mutant mice exhibit upregulated creatine kinase. • Isothermal titration calorimetry detected creatine kinase/α-crystallin interaction. • The protein-protein interaction is thermodynamically favorable and entropy driven. |
| Databáze: | OpenAIRE |
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