Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100.
Bottom Line: Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known.Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions.Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100.
Affiliation: Degenerative Diseases Research Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.Show MeSH
Mentions: To study the functional role of PDI1 in hepatic VLDL assembly, we first generated adenoviruses expressing Pdi1 short hairpin RNAs (shRNAs) to knock down PDI1 in rodent hepatoma cells. Adenovirus infects hepatoma cells with high efficiency, ensuring effective knockdown of target genes. Accordingly, Pdi1 mRNA levels were decreased by >95%, and PDI1 protein levels were almost undetectable in Hepa1-6 cells 3 d after adenoviral infection (confirmed with two sets of shRNAs; Supplemental Figure S1, A and B). For the purposes of this article, all studies were conducted with shRNA 1. Adenovirus-mediated Pdi1 knockdown was then tested in McA-RH7777 (McA) rat hepatoma cells, a widely used model for studying hepatic lipoprotein secretion (Boren et al., 1994; Liang et al., 2004; Yamaguchi et al., 2003). McA cells secrete predominantly apoB100 in VLDL particles with size and density distribution similar to that of human hepatocytes (Boren et al., 1994). Pdi1 mRNA and protein levels were decreased ∼80% after shRNA-mediated knockdown in McA cells (Figure 1, A and B). Moreover, knockdown of Pdi1 did not lead to compensatory up-regulation or off-target knockdown of other PDI family members, including ERp57 and ERp72 (Figure 1A and Supplemental Figure S1A) in both Hepa1-6 and McA cells. In addition, the Pdi1 knockdown mediated by adenovirus persisted at least 8 d after infection, allowing adequate time to perform the functional studies in Pdi1-knockdown cells (Supplemental Figure S2). These findings gave us confidence that our approach allows us to examine the phenotypes associated with Pdi1 knockdown in McA cells.
Affiliation: Degenerative Diseases Research Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.