Inhibition of Cu²⁺- mediated generation of reactive oxygen species by the small heat shock protein αβ-crystallin: The relative contributions of the N- and C-terminal domains

Abstract

Oxidative stress, Cu²⁺ homeostasis, and small heat shock proteins (sHsp's) have important implications in several neurodegenerative diseases. The ubiquitous sHsp aB-crystallin is an oligomeric protein that binds Cu²⁺. We have investigated the relative contributions of the N- and C-terminal (C-TDαβ-crystallin) domains of αβ-crystallin to its Cu²⁺-binding and redox-attenuation properties and mapped the Cu²⁺-binding regions. C-TDα β-crystallin binds Cu²⁺ with slightly less affinity and inhibits Cu²⁺-catalyzed, ascorbate-mediated generation of ROS to a lesser extent than αβ -crystallin. [Cu²⁺]/[subunit] stoichiometries for redox attenuation by αβ-crystallin and C-TDαβ-crystallin are 5 and 2, respectively. Both αβ-crystallin and C-TDαβ-crystallin also inhibit the Fenton reaction of hydroxyl radical formation. Trypsinization of αβ -crystallin bound to a Cu²⁺–NTA column and MALDI-TOF analysis of column-bound peptides yielded three peptides located in the N-terminal domain, and in-solution trypsinization of aB-crystallin followed by Cu²⁺–NTA column chromatography identified four additional Cu²⁺-binding peptides located in the C-terminal domain. Thus, Cu²⁺-binding regions are distributed in the N- and C-terminal domains. Small-angle X-ray scattering and sedimentation-velocity measurements indicate quaternary structural changes in αβ-crystallin upon Cu²⁺ binding. Our study indicates that an oligomer of αβ-crystallin can sequester a large number (∼ 150) of Cu²⁺ ions. It acts like a “Cu²⁺ sponge,” exhibits redox attenuation of Cu²⁺, and has potential roles in Cu²⁺ homeostasis and in preventing oxidative stress.