title: Ubiquitin-dependent lysosomal degradation of the HNE-modified proteins in lens epithelial cells.
abstract: 4-hydroxynonenal (HNE), a highly reactive lipid peroxidation product, may adversely modify proteins. Accumulation of HNE-modified proteins may be responsible for pathological lesions associated with oxidative stress. The objective of this work was to determine how HNE-modified proteins are removed from cells. The data showed that alphaB-crystallin modified by HNE was ubiquitinated at a faster rate than that of native alphaB-crystallin in a cell-free system. However, its susceptibility to proteasome-dependent degradation in the cell-free system did not increase. When delivered into cultured lens epithelial cells, HNE-modified alphaB-crystallin was degraded at a faster rate than that of unmodified alphaB-crystallin. Inhibition of the lysosomal activity stabilized HNE-modified alphaB-crystallin, but inhibition of the proteasome activity alone had little effect. To determine if other HNE-modified proteins are also degraded in a ubiquitin-dependent lysosomal pathway, lens epithelial cells were treated with HNE and the removal of HNE-modified proteins in the cells was monitored. The levels of HNE-modified proteins in the cell decreased rapidly upon removal of HNE from the medium. Depletion of ATP or the presence of MG132, a proteasome/lysosome inhibitor, resulted in stabilization of HNE-modified proteins. However, proteasome-specific inhibitors, lactacystin-beta-lactone and epoxomicin, could not stabilize HNE-modified proteins in the cells. In contrast, chloroquine, a lysosome inhibitor, stabilized HNE-modified proteins. The enrichment of HNE-modified proteins in the fraction of ubiquitin conjugates suggests that HNE-modified proteins are preferentially ubiquitinated. Taken together, these findings show that HNE-modified proteins are degraded via a novel ubiquitin and lysosomal-dependent but proteasome-independent pathway.
authors.affiliation: Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111, USA.
authors.forename: Carla
authors.initials: C
authors.lastname: Marques
authors.forename: Paulo
authors.initials: P
authors.lastname: Pereira
authors.forename: Allen
authors.initials: A
authors.lastname: Taylor
authors.forename: Jack N
authors.initials: JN
authors.lastname: Liang
authors.forename: Venkat N
authors.initials: VN
authors.lastname: Reddy
authors.forename: Luke I
authors.initials: LI
authors.lastname: Szweda
authors.forename: Fu
authors.initials: F
authors.lastname: Shang
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000447:Aldehydes / Q000494:pharmacology*; D002474:Cell-Free System; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D018384:Oxidative Stress; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D025801:Ubiquitin / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D007976:Leupeptins; D009842:Oligopeptides; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D038203:alpha-Crystallin B Chain; C488643:lactacystin beta-lactone; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; C027576:4-hydroxy-2-nonenal; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine; C078846:epoxomicin