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.

Keywords: 4-HNE; 4-ONE; Alcoholic liver disease; Electrophile; Free radicals; HSP; Protein oxidation.

Figure 1. GRP78 is a target for modification by 4-HNE and 4-ONE in a murine model for early-stage ALD

Positive immunostaining was observed following western blotting with antibodies directed against 4-HNE-modified proteins (A) and 4-ONE-modified proteins (B). As a reference, immunostaining was also conducted with antibodies directed against the C-terminal KDEL sequence of GRP78 (C) Coomassie blue staining reveals GRP78, which was positively identified utilizing MALDI-MS (D). GRP78 is indicated by the arrow in each figure.

Figure 2. Carbonylation of GRP78 is increased following sustained ethanol consumption

(A) Carbonylated GRP78 is visualized through biotin hydrazide conjugation, streptavidin pulldown and GRP78 western blotting. While the expression of GRP78 does not change following ethanol consumption, carbonylated protein is increased. (B) Blot densitometry of (A) reveals a statistically significant increase in modified protein.

Figure 3. Adduction by 4-HNE and 4-ONE leads to proteins-protein cross-linking

A.) Western blotting of purified GRP78 following adduction with either 4-HNE or 4-ONE. Adduction induces high molecular weight protein-protein cross-linking with purified GRP78 at physiologically relevant doses of either 4-HNE (B) or 4-ONE (C). Chemical stabilization of these adducts using NaBH₄, displays the high susceptibility of this lysine cross-linking with concentrations as low as 3μM (1x).

Figure 4. 4-HNE and 4-ONE adduction of GRP78 results in a concentration-dependent decrease in ATP hydrolysis

Treatment of GRP78 with 4-HNE (open bars) or 4-ONE (closed bars) reveals a corresponding decrease in ATPase activity. Estimated IC50 values were calculated revealing a Ki of 51.8μM for 4-HNE and a Ki of 43.9μM for 4-ONE.

Figure 5. Chaperone-mediated refolding of CS and MDH is preserved following adduction

GRP78-mediated refolding of (A) MDH or (B) CS is unaffected by electrophile adduction with either 4-HNE (closed bars) or 4-ONE (open bars). (C) Sequence alignment using ClustalW demonstrates a conserved Cys residue in electrophile-susceptible HSPs. HSPs lacking this Cys residue are presented in green font. Mean is plotted with error bars representing SEM.

Figure 6. Computational-based molecular modeling of 4-ONE adduction reveals potential conformational changes in the ATPase domain of GRP78

Multiple viewpoints of the overlay of energy-minimized native (blue) and 4-ONE adducted (magenta) human GRP78. 4-ONE adducts are depicted as yellow sticks and the specific identities of the modified residues indicated. Key conformational changes in the ATP binding site between the native (blue, left inset) and 4-ONE adducted (magenta, right inset) are shown. Solid orange lines indicate predicted pi bond interactions and dashed green lines indicate predicted hydrogen bond interactions.

Figure 7. Electrophilic modification of GRP78 decreases its affinity for ATP

(A) Treatment of GRP78 with 4-HNE (top) or 4-ONE (bottom) results in a concentration-dependent decrease in ATP binding (B) Quantification of (A); mean is plotted from an N of 3; error bars represent SEM.

Figure 8

Although ATPase activity is inhibited, the chaperone function of GRP78 is resistant to the deleterious impact of electrophile adduction.