Inorganic Chemistry

Copper reductase activity and free radical chemistry by cataract-associated human lens γ-crystallins

Authors

  • Giovanni Palomino-Vizcaino Department of Chemistry, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, Mexico ,
  • Jose A. Domínguez-Calva Department of Chemistry, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, Mexico ,
  • Nils Schuth Department of Chemistry, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, Mexico ,
  • Eduardo Martínez-Jurado Department of Chemistry, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, Mexico ,
  • Oscar Rodríguez-Meza Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico ,
  • Eugene Serebryany Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA ,
  • Jonathan A. King Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA ,
  • Thomas Kroll Stanford Synchrotron Radiation Laboratory (SSRL), SLAC National Accelerator Laboratory, Menlo Park, CA, USA. ,
  • Miguel Costas Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico ,
  • Liliana Quintanar Department of Chemistry, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, Mexico

Abstract

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 Cu2+ ions with γD-, γC-, and γS- crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, SDS-PAGE, circular dichroism and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: i) loss of beta-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. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated reduction of γ-crystallin-bound Cu2+ 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’ Cu2+ 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 be acting, not only as structural proteins, but also as key players for metal and redox homeostasis.

Content

Thumbnail image of CuCrystallin_Manuscript_6Dec22.pdf

Supplementary material

Thumbnail image of CuCrystallin_SupportingInformation_27Nov22.pdf
Supporting Information
Supporting information includes: experimental section; calorimetry modeling details; scan rate dependance of Tm and DSC thermograms for γ-crystallins; turbidity assays of γ-crystallins with Cu in the pres-ence of B-crystallin; turbidity assays, CD spectra and thermal dena-turation curves for the separate N- and C- terminal domains of γ-crystallins in the presence of different amounts of Cu ions; DSC ther-mal denaturation curves and Arrhenius plots for HγD and its N- and C- terminal domains with and without Cu2+ ions; Peisach-Blumberg correlations for Az and gz values for Cu2+ complexes; comparison of XANES and EXAFS for the two Cu+ species observed in γ-crystallins; tables listing Tm values for all proteins reported here; and table listing dihedral angles for all Tyr residues in the crystal structure (PDB 1HK0) of HγD crystallin.