title: Degradation of C-terminal truncated alpha A-crystallins by the ubiquitin-proteasome pathway.
abstract: OBJECTIVE
  Calpain-mediated C-terminal cleavage of alpha A-crystallins occurs during aging and cataractogenesis. The objective of the present study was to explore the role of the ubiquitin-proteasome pathway (UPP) in degrading C-terminal truncated alpha A-crystallins.
  
  
  METHODS
  Recombinant wild-type (wt) alpha A-crystallin and C-terminal truncated alpha A(1-168)-, alpha A(1-163)-, and alpha A(1-162)-crystallins were expressed in Escherichia coli and purified to homogeneity. The wt and truncated alpha A-crystallins were labeled with (125)I, and proteolytic degradation was determined using both lens fiber lysate and reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. Far UV circular dichroism, tryptophan fluorescence intensity, and binding to the hydrophobic fluorescence probe Bis-ANS were used to characterize the wt and truncated alpha A-crystallins. Oligomer sizes of these crystallins were determined by multiangle light-scattering.
  
  
  RESULTS
  Whereas wt alpha A-crystallin was degraded moderately in both lens fiber and reticulocyte lysates, alpha A(1-168)-crystallin was resistant to degradation. The susceptibility of alpha A(1-163)-crystallin to degradation was comparable to that of wt alpha A-crystallin. However, alpha A(1-162)-crystallin was much more susceptible than wt alpha A-crystallin to degradation in both lens fiber and reticulocyte lysates. The degradation of both wt and C-terminal truncated alpha A(1-162)-crystallins requires adenosine triphosphate (ATP) and was stimulated by addition of a ubiquitin-conjugating enzyme, Ubc4. The degradation was substantially inhibited by the proteasome inhibitor MG132 and a dominant negative mutant of ubiquitin, K6W-Ub, indicating that at least part of the proteolysis was mediated by the UPP. Spectroscopic analyses of wt and C-terminal truncated alpha A-crystallins revealed that C-terminal truncation of alpha A-crystallin resulted in only subtle changes in secondary structures. However, C-terminal truncations resulted in significant changes in surface hydrophobicity and thermal stability. Thus, these conformational changes may reveal or mask the signals for the ubiquitin-dependent degradation.
  
  
  CONCLUSIONS
  The present data demonstrate that C-terminal cleavage of alpha A-crystallin not only alters its conformation and thermal stability, but also its susceptibility to degradation by the UPP. The rapid degradation of alpha A(1-162) by the UPP may prevent its accumulation in the lens.
authors.affiliation: Laboratory for Nutrition and Vision Research, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111, USA.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Zhang
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Dudek
authors.forename: Bingfen
authors.initials: B
authors.lastname: Liu
authors.forename: Linlin
authors.initials: L
authors.lastname: Ding
authors.forename: Alexandre F
authors.initials: AF
authors.lastname: Fernandes
authors.forename: Jack J
authors.initials: JJ
authors.lastname: Liang
authors.forename: Joseph
authors.initials: J
authors.lastname: Horwitz
authors.forename: Allen
authors.initials: A
authors.lastname: Taylor
authors.forename: Fu
authors.initials: F
authors.lastname: Shang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D007908:Lens, Crystalline / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D025801:Ubiquitin / Q000502:physiology*; D043743:Ubiquitin-Protein Ligase Complexes / Q000502:physiology*; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000378:metabolism*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; D025801:Ubiquitin; D038202:alpha-Crystallin A Chain; D043743:Ubiquitin-Protein Ligase Complexes; D046988:Proteasome Endopeptidase Complex