Copper Reductase Activity and Free Radical Chemistry by Cataract-Associated Human Lens γ-Crystallins

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, 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 Cu2+ binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated the 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 act not only as structural proteins but also as key players for metal and redox homeostasis.

ATCUN-like Copper Site in βB2-Crystallin Plays a Protective Role in Cataract-Associated Aggregation

“…Copper-induced aggregation of βB2-crystallin seems to involve several mechanisms including metal-bridging, disulfide-bridging, loss of protein stability, and copper reduction. This complex scenario is partially similar to that observed for copper-induced aggregation of human γ-crystallins, 31 where the mechanism involves: copper coordination, unfolding of the protein, intermolecular oligomerization through disulfide and metal-bridging, and copper redox chemistry that leads to a tyrosine-based radical. No protein-based radical is observed in the case of βB2-crystallin (Figure S14), while all other mechanisms are at work.…”

“…A previous study of γ-crystallins shows that Cu 2+ coordination to these proteins can lead to reduction of the metal ion under aerobic conditions . Here, we have used EPR and X-ray absorption/emission spectroscopy (XAS/XES) to probe if the Cu 2+ binding sites in βB2-crystallin are redox-active (Figure ).…”

“…Turbidity assays were performed as described previously . In summary, βB2-crystallin ad its N-truncated version were added to a 96-well microplate independently, preincubated for 10 min at 37 °C, and then incubated again incubated with or without metal ions (50 μM, 200 μL final protein concentration and volume, in 10 mM MOPS, 50 mM NaCl, pH 7 buffer); then, after 30 min of the addition of metal ions, 100 equiv of EDTA (or an equivalent volume of buffer for non-chelating condition) were added.…”

“…A previous study of γ-crystallins shows that Cu 2+ coordination to these proteins can lead to reduction of the metal ion under aerobic conditions. 31 Here, we have used EPR and X-ray absorption/ emission spectroscopy (XAS/XES) to probe if the Cu 2+ binding sites in βB2-crystallin are redox-active (Figure 7). These experiments were performed with 1 equiv of Cu 2+ ions; under these conditions, Mode 1 and Mode 2 are observed for the βB2crystallin WT, while Mode 2 is predominantly observed for the N-truncated protein by EPR (Figure 7A).…”

“…Turbidity assays were performed as described previously. 31 In summary, βB2-crystallin ad its N-truncated version were added to a 96-well microplate independently, preincubated for 10 min at 37 °C, and then incubated again incubated with or without metal ions ( The protein aggregates at the endpoint of the turbidity essay were collected and separated by centrifugation into soluble and insoluble fractions. An equivalent volume of Laemmli buffer (with or without 2mercaptoethanol) was added to each sample and boiled for 10 min.…”