Dataset Information

Hydrophobic core mutations associated with cataract development in mice destabilize human gammaD-crystallin.

ABSTRACT: The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human gammaD-crystallin (HgammaD-Crys) is a monomeric, two-domain protein of the lens central nucleus. Both domains of this long lived protein have double Greek key beta-sheet folds with well packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the gamma-crystallin buried cores (two in the N-terminal domain (N-td) and one in the C-terminal domain (C-td)) cause early onset cataract in mice, presumably an aggregated state of the mutant crystallins. It has not been possible to identify the aggregating precursor within lens tissues. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of gamma-crystallins, mouse mutant substitutions were introduced into HgammaD-Crys. The mutant proteins L5S, V75D, and I90F were expressed and purified from Escherichia coli. WT HgammaD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded and the C-td native-like. L5S and V75D in the N-td also displayed three-state unfolding transitions, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. I90F destabilized the C-td, shifting the overall unfolding transition to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments showed that the N-tds of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded more rapidly. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.

SUBMITTER: Moreau KL

PROVIDER: S-EPMC2785171 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Hydrophobic core mutations associated with cataract development in mice destabilize human gammaD-crystallin.

Moreau Kate L KL King Jonathan J

The Journal of biological chemistry 20090916 48

The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human gammaD-crystallin (HgammaD-Crys) is a monomeric, two-domain protein of the lens central nucleus. Both domains of this long lived protein have double Greek key beta-sheet folds with well packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the gamma-crystallin buried cores (two in the N-terminal domain (N-td) and one in the C-terminal domain (C-td)) cause early onset cataract ...[more]

PMID: 19758984

Similar Datasets

Project description: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 ?A-crystallin (cryaa) and ?B-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 ?-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 ?-crystallin, we investigated whether ?-crystallin interacts with and forms complexes with CK, in vitro. Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 ?-crystallin subunits, with a Kd of 3.3 × 10-7 M, and that the interaction between ?-crystallin and CK was endothermic, thermodynamically favorable, and entropy-driven. High-salt concentrations did not affect the interaction between CK and ?-crystallin, suggesting that the interaction between CK and ?-crystallin is primarily hydrophobic. Gel permeation chromatography (GPC) detected water-soluble ?-crystallin and CK complexes, as determined by increased light scattering after complex formation. In addition, CK and ?-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 ?-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.

Dataset Information

Hydrophobic core mutations associated with cataract development in mice destabilize human gammaD-crystallin.

Publications

Hydrophobic core mutations associated with cataract development in mice destabilize human gammaD-crystallin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets