title: Expression of K6W-ubiquitin in lens epithelial cells leads to upregulation of a broad spectrum of molecular chaperones.
abstract: OBJECTIVE
  Accumulation and precipitation of abnormal proteins are associated with many age-related diseases. The ubiquitin-proteasome pathway (UPP) is one of the protein quality control mechanisms that selectively degrade damaged or obsolete proteins. The other arm of the protein quality control mechanism is molecular chaperones, which bind to and help refold unfolded or misfolded proteins. We previously showed that the molecular chaperones and the UPP work in a competitive manner in eliminating the denatured proteins. To further investigate the interaction between the two protein quality control mechanisms, we determined the effects of the impairment of the UPP on the expression of molecular chaperones in human lens epithelial cells (HLEC).
  
  
  METHODS
  K6W-ubiquitin, a dominant negative inhibitor of the UPP, was expressed in confluent HLEC via an adenoviral vector. The mRNA levels of cytoplasmic and endoplasmic reticulum (ER) chaperones were determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Protein levels for these chaperones were determined by western blotting.
  
  
  RESULTS
  Expression of K6W-ubiquitin in HLEC increased the expression of a broad spectrum of molecular chaperones. Among the heat-shock proteins, mRNA for alphaB-crystallin, Hsp70, and Hsp90 increased 27 fold, 21 fold, and twofold, respectively, in response to K6W-ubiquitin expression. Among the ER chaperones and ER stress related factors, mRNA levels of protein disulfide isomerase, Grp75, Grp78, Grp94, and the CAAT/enhancer binding protein homologous protein (CHOP) increased from 1.7 fold to 3.7 fold. The mRNA for Hsp60 also increased 1.6 fold in response to the expression of K6W-ubiquitin. The expression pattern of these chaperones in response to the expression of K6W ubiquitin is similar to that obtained when cells were treated with proteasome inhibitors or heat-shock.
  
  
  CONCLUSIONS
  It appears that the upregulation of these chaperones is related to the elevated levels of abnormal proteins in the cells. These findings support our hypothesis that the molecular chaperones and the UPP may back each other up in the process of protein quality control. The upregulation of molecular chaperones in response to the expression of a dominant negative ubiquitin may compensate for the impairment of the UPP in the degradation of abnormal proteins.
authors.affiliation: USDA HNRCA at Tufts University, Boston, Massachusetts, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Bian
authors.forename: A F
authors.initials: AF
authors.lastname: Fernandes
authors.forename: A
authors.initials: A
authors.lastname: Taylor
authors.forename: M
authors.initials: M
authors.lastname: Wu
authors.forename: P
authors.initials: P
authors.lastname: Pereira
authors.forename: F
authors.initials: F
authors.lastname: Shang
mesh_terms: D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D000091342:Endoplasmic Reticulum Chaperone BiP; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018869:Heat-Shock Response / Q000187:drug effects; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D061988:Proteasome Inhibitors; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics*
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D007976:Leupeptins; D018832:Molecular Chaperones; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D025801:Ubiquitin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde