title: Inhibition of Cu2+-mediated generation of reactive oxygen species by the small heat shock protein αB-crystallin: the relative contributions of the N- and C-terminal domains.
abstract: Oxidative stress, Cu(2+) homeostasis, and small heat shock proteins (sHsp's) have important implications in several neurodegenerative diseases. The ubiquitous sHsp αB-crystallin is an oligomeric protein that binds Cu(2+). We have investigated the relative contributions of the N- and C-terminal (C-TDαB-crystallin) domains of αB-crystallin to its Cu(2+)-binding and redox-attenuation properties and mapped the Cu(2+)-binding regions. C-TDαB-crystallin binds Cu(2+) with slightly less affinity and inhibits Cu(2+)-catalyzed, ascorbate-mediated generation of ROS to a lesser extent than αB-crystallin. [Cu(2+)]/[subunit] stoichiometries for redox attenuation by αB-crystallin and C-TDαB-crystallin are 5 and 2, respectively. Both αB-crystallin and C-TDαB-crystallin also inhibit the Fenton reaction of hydroxyl radical formation. Trypsinization of αB-crystallin bound to a Cu(2+)-NTA column and MALDI-TOF analysis of column-bound peptides yielded three peptides located in the N-terminal domain, and in-solution trypsinization of αB-crystallin followed by Cu(2+)-NTA column chromatography identified four additional Cu(2+)-binding peptides located in the C-terminal domain. Thus, Cu(2+)-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 αB-crystallin upon Cu(2+) binding. Our study indicates that an oligomer of αB-crystallin can sequester a large number (~150) of Cu(2+) ions. It acts like a "Cu(2+) sponge," exhibits redox attenuation of Cu(2+), and has potential roles in Cu(2+) homeostasis and in preventing oxidative stress.
authors.affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad 500 007, India.
authors.forename: Sankaralingam
authors.initials: S
authors.lastname: Prabhu
authors.forename: Volety
authors.initials: V
authors.lastname: Srinivas
authors.forename: Tangirala
authors.initials: T
authors.lastname: Ramakrishna
authors.forename: Bakthisaran
authors.initials: B
authors.lastname: Raman
authors.forename: Ch Mohan
authors.initials: ChM
authors.lastname: Rao
mesh_terms: D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D050888:Heat-Shock Proteins, Small / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019636:Neurodegenerative Diseases / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary / Q000235:genetics; D017382:Reactive Oxygen Species / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D050888:Heat-Shock Proteins, Small; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D038203:alpha-Crystallin B Chain; D003300:Copper