title: Oxidative stress-mediated aldehyde adduction of GRP78 in a mouse model of alcoholic liver disease: functional independence of ATPase activity and chaperone function.
abstract: Pathogenesis in alcoholic liver disease (ALD) is complicated and multifactorial but clearly involves oxidative stress and inflammation. Currently, conflicting reports exist regarding the role of endoplasmic reticulum (ER) stress in the etiology of ALD. The glucose-regulated protein 78 (GRP78) is the ER homolog of HSP70 and plays a critical role in the cellular response to ER stress by serving as a chaperone assisting protein folding and by regulating the signaling of the unfolded protein response (UPR). Comprising three functional domains, an ATPase, a peptide-binding, and a lid domain, GRP78 folds nascent polypeptides via the substrate-binding domain. Earlier work has indicated that the ATPase function of GRP78 is intrinsically linked and essential to its chaperone activity. Previous work in our laboratory has indicated that GRP78 and the UPR are not induced in a mouse model of ALD but that GRP78 is adducted by the lipid electrophiles 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) in vivo. As impairment of GRP78 has the potential to contribute to pathogenesis in ALD, we investigated the functional consequences of aldehyde adduction on GRP78 function. Identification of 4-HNE and 4-ONE target residues in purified human GRP78 revealed a marked propensity for Lys and His adduction within the ATPase domain and a relative paucity of adduct formation within the peptide-binding domain. Consistent with these findings, we observed a concomitant dose-dependent decrease in ATP-binding and ATPase activity without any discernible impairment of chaperone function. Collectively, our data indicate that ATPase activity is not essential for GRP78-mediated chaperone activity and is consistent with the hypothesis that ER stress does not play a primary initiating role in the early stages of ALD.
authors.affiliation: Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Galligan
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
authors.forename: Kristofer S
authors.initials: KS
authors.lastname: Fritz
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
authors.forename: Donald S
authors.initials: DS
authors.lastname: Backos
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
authors.forename: Colin T
authors.initials: CT
authors.lastname: Shearn
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Smathers
authors.affiliation: Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: MacLean
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
authors.forename: Philip R
authors.initials: PR
authors.lastname: Reigan
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address: Dennis.Petersen@UCDenver.edu.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Petersen
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000447:Aldehydes / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003198:Computer Simulation; D004721:Endoplasmic Reticulum / Q000473:pathology; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000473:pathology; D008108:Liver Diseases, Alcoholic / Q000209:etiology / Q000473:pathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D018384:Oxidative Stress; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D056811:Unfolded Protein Response
chemical_list: C403894:4-oxo-2-nonenal; D000447:Aldehydes; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D000251:Adenosine Triphosphatases; C027576:4-hydroxy-2-nonenal
keywords: 4-HNE; 4-ONE; Alcoholic liver disease; Electrophile; Free radicals; HSP; Protein oxidation