title: Formation of amyloid fibrils in vitro from partially unfolded intermediates of human gammaC-crystallin.
abstract: OBJECTIVE
  Mature-onset cataract results from the formation of light-scattering aggregates of lens crystallins. Although oxidative or mutational damage may be a prerequisite, little is known of the initiation or nucleation of these aggregated states. In mice carrying mutations in gamma-crystallin genes, a truncated form of gamma-crystallin formed intranuclear filamentous inclusions within lens fiber cells. Previous studies have shown that bovine crystallins and human gammaD-crystallin form amyloid fibrils under denaturing conditions in vitro. The amyloid fibril formation of human gammaC-crystallin (HgammaC-Crys) induced by low pH, together with characterization of a partially unfolded intermediate in the process were investigated.
  
  
  METHODS
  HgammaC-Crys was expressed and purified from Escherichia coli. Partially unfolded intermediates were detected by tryptophan fluorescence spectroscopy and UV resonance Raman spectroscopy. The aggregation into amyloid fibrils was monitored by solution turbidity and fluorescence assay. The morphology of aggregates was characterized using transmission electron microscopy (TEM). Secondary structure of the peptides in their fibrillar state was characterized using Fourier transform infrared spectroscopy (FTIR).
  
  
  RESULTS
  The structure of HgammaC-Crys was perturbed at low pH. Partially unfolded intermediates were detected when solution pH was lowered to pH 3. At pH 3, HgammaC-Crys aggregated into amyloid fibrils. The kinetics and extent of the reaction was dependent on protein concentration, pH, and temperature. TEM images of aggregates revealed aggregation stages from short to long fibrils and from long fibrils to light-scattering fibril networks. FTIR spectroscopy confirmed the cross-beta character of the secondary structure of these fibrils.
  
  
  CONCLUSIONS
  HgammaC-Crys formed amyloid fibrils on incubation at low pH via a partially unfolded intermediate. This process could contribute to the early stages of the formation of light-scattering species in the eye lens.
authors.affiliation: Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China. ytwang@sjtu.edu.cn
authors.forename: Yongting
authors.initials: Y
authors.lastname: Wang
authors.forename: Sarah
authors.initials: S
authors.lastname: Petty
authors.forename: Amy
authors.initials: A
authors.lastname: Trojanowski
authors.forename: Kelly
authors.initials: K
authors.lastname: Knee
authors.forename: Daniel
authors.initials: D
authors.lastname: Goulet
authors.forename: Ishita
authors.initials: I
authors.lastname: Mukerji
authors.forename: Jonathan
authors.initials: J
authors.lastname: King
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000737:chemistry; D038222:gamma-Crystallins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000682:Amyloid; C522806:Crygc protein, human; D038222:gamma-Crystallins; D014364:Tryptophan
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