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Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100.

Wang S, Park S, Kodali VK, Han J, Yip T, Chen Z, Davidson NO, Kaufman RJ - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Degenerative Diseases Research Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.

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Knockdown of Pdi1 decreases apoB100 lipidation in McA cells. (A–D) Pdi1 knockdown reduces apoB100 lipidation. McA cells were continuously labeled with [35S]Met/Cys containing BSA (A) or 400 μM OA (C) for 4 h. The medium was collected for determination of buoyant density of secreted apoB-containing lipoproteins from control (NSi) and Pdi1 knockdown (PDI1i) McA cells by DGUC, followed by immunoprecipitation for apoB100. The amount of apoB100 in each fraction was quantified by ImageJ. Relative distributions were calculated according to the percentage of apoB100 in each fraction relative to its total intensity across the gel (B, D). (E, F) Efficient TG secretion requires catalytically active PDI1. TG secretion (E) and MTP activity (F) were decreased in Pdi1-knockdown McA cells. TG synthesis and secretion (E) and MTP activity (F) were measured in control and Pdi1-knockdown McA cells. *p < 0.01 vs. NSi. (G–I) Overexpression of human wild-type PDI1 but not catalytically inactive PDI1 rescues decreased TG secretion in Pdi1-knockdown McA cells. (G) Wild-type and mutant PDI1 are expressed at equal levels in Pdi1-knockdown McA cells. β-Galactosidase (β-gal), human wild-type PDI1 (PDI), and catalytically inactive PDI1 (PDImu) were expressed using adenoviruses, and their expression levels were measured by Western blotting analysis. TG synthesis and secretion (H) and MTP activity (I) were measured in control cells or Pdi1-knockdown cells with overexpression of β-gal, PDI, or PDImu McA cells. PBS denotes no adenovirus infection. *p < 0.01 vs. PBS, PDI1, or PDI1mu; **p < 0.01 vs. PBS or PDI1.
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Figure 3: Knockdown of Pdi1 decreases apoB100 lipidation in McA cells. (A–D) Pdi1 knockdown reduces apoB100 lipidation. McA cells were continuously labeled with [35S]Met/Cys containing BSA (A) or 400 μM OA (C) for 4 h. The medium was collected for determination of buoyant density of secreted apoB-containing lipoproteins from control (NSi) and Pdi1 knockdown (PDI1i) McA cells by DGUC, followed by immunoprecipitation for apoB100. The amount of apoB100 in each fraction was quantified by ImageJ. Relative distributions were calculated according to the percentage of apoB100 in each fraction relative to its total intensity across the gel (B, D). (E, F) Efficient TG secretion requires catalytically active PDI1. TG secretion (E) and MTP activity (F) were decreased in Pdi1-knockdown McA cells. TG synthesis and secretion (E) and MTP activity (F) were measured in control and Pdi1-knockdown McA cells. *p < 0.01 vs. NSi. (G–I) Overexpression of human wild-type PDI1 but not catalytically inactive PDI1 rescues decreased TG secretion in Pdi1-knockdown McA cells. (G) Wild-type and mutant PDI1 are expressed at equal levels in Pdi1-knockdown McA cells. β-Galactosidase (β-gal), human wild-type PDI1 (PDI), and catalytically inactive PDI1 (PDImu) were expressed using adenoviruses, and their expression levels were measured by Western blotting analysis. TG synthesis and secretion (H) and MTP activity (I) were measured in control cells or Pdi1-knockdown cells with overexpression of β-gal, PDI, or PDImu McA cells. PBS denotes no adenovirus infection. *p < 0.01 vs. PBS, PDI1, or PDI1mu; **p < 0.01 vs. PBS or PDI1.

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.


Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100.

Wang S, Park S, Kodali VK, Han J, Yip T, Chen Z, Davidson NO, Kaufman RJ - Mol. Biol. Cell (2014)

Knockdown of Pdi1 decreases apoB100 lipidation in McA cells. (A–D) Pdi1 knockdown reduces apoB100 lipidation. McA cells were continuously labeled with [35S]Met/Cys containing BSA (A) or 400 μM OA (C) for 4 h. The medium was collected for determination of buoyant density of secreted apoB-containing lipoproteins from control (NSi) and Pdi1 knockdown (PDI1i) McA cells by DGUC, followed by immunoprecipitation for apoB100. The amount of apoB100 in each fraction was quantified by ImageJ. Relative distributions were calculated according to the percentage of apoB100 in each fraction relative to its total intensity across the gel (B, D). (E, F) Efficient TG secretion requires catalytically active PDI1. TG secretion (E) and MTP activity (F) were decreased in Pdi1-knockdown McA cells. TG synthesis and secretion (E) and MTP activity (F) were measured in control and Pdi1-knockdown McA cells. *p < 0.01 vs. NSi. (G–I) Overexpression of human wild-type PDI1 but not catalytically inactive PDI1 rescues decreased TG secretion in Pdi1-knockdown McA cells. (G) Wild-type and mutant PDI1 are expressed at equal levels in Pdi1-knockdown McA cells. β-Galactosidase (β-gal), human wild-type PDI1 (PDI), and catalytically inactive PDI1 (PDImu) were expressed using adenoviruses, and their expression levels were measured by Western blotting analysis. TG synthesis and secretion (H) and MTP activity (I) were measured in control cells or Pdi1-knockdown cells with overexpression of β-gal, PDI, or PDImu McA cells. PBS denotes no adenovirus infection. *p < 0.01 vs. PBS, PDI1, or PDI1mu; **p < 0.01 vs. PBS or PDI1.
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Figure 3: Knockdown of Pdi1 decreases apoB100 lipidation in McA cells. (A–D) Pdi1 knockdown reduces apoB100 lipidation. McA cells were continuously labeled with [35S]Met/Cys containing BSA (A) or 400 μM OA (C) for 4 h. The medium was collected for determination of buoyant density of secreted apoB-containing lipoproteins from control (NSi) and Pdi1 knockdown (PDI1i) McA cells by DGUC, followed by immunoprecipitation for apoB100. The amount of apoB100 in each fraction was quantified by ImageJ. Relative distributions were calculated according to the percentage of apoB100 in each fraction relative to its total intensity across the gel (B, D). (E, F) Efficient TG secretion requires catalytically active PDI1. TG secretion (E) and MTP activity (F) were decreased in Pdi1-knockdown McA cells. TG synthesis and secretion (E) and MTP activity (F) were measured in control and Pdi1-knockdown McA cells. *p < 0.01 vs. NSi. (G–I) Overexpression of human wild-type PDI1 but not catalytically inactive PDI1 rescues decreased TG secretion in Pdi1-knockdown McA cells. (G) Wild-type and mutant PDI1 are expressed at equal levels in Pdi1-knockdown McA cells. β-Galactosidase (β-gal), human wild-type PDI1 (PDI), and catalytically inactive PDI1 (PDImu) were expressed using adenoviruses, and their expression levels were measured by Western blotting analysis. TG synthesis and secretion (H) and MTP activity (I) were measured in control cells or Pdi1-knockdown cells with overexpression of β-gal, PDI, or PDImu McA cells. PBS denotes no adenovirus infection. *p < 0.01 vs. PBS, PDI1, or PDI1mu; **p < 0.01 vs. PBS or PDI1.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Degenerative Diseases Research Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.

Show MeSH
Related in: MedlinePlus