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.Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells.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
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Mentions: Poorly lipidated nascent apoB100 is readily degraded cotranslationally in a proteasome-dependent and/or -independent manner (Pan et al., 2008). To determine whether Pdi1 knockdown disrupts lipidation of apoB, we analyzed the density distribution of apoB100 particles secreted from control and Pdi1-knockdown cells. Cells were labeled continuously for 3 h with [35S]Met/Cys, and the labeled medium was subjected to density gradient ultracentrifugation (DGUC) to separate apoB100-containing particles based on their relative hydrated density. ApoB100-containing particles secreted from control cells migrated primarily in the VLDL fractions (fractions 1 and 2; Figure 3, A and B) either in the presence or absence of oleic acid in the media. In contrast, most of the apoB100-containing particles secreted from Pdi1 knockdown cells were distributed as low-density lipoproteins (fractions 3–6; Figure 3, A and B). We interpret this shift to suggest that the relative distribution of apoB100 in the lipid-rich VLDL fractions from Pdi1-knockdown cells was decreased, whereas the proportion of apoB100 migrating in lipid-poor particles correspondingly increased. In contrast to apoB100, apoB48 was mainly associated with the lipid-poor particles and was not altered by Pdi1 knockdown in McA cells (Figure 3, A and B). This result indicates that PDI1 is required for full lipidation of apoB100 and VLDL maturation.
Affiliation: Degenerative Diseases Research Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.