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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 sensitizes cells to ER stress. (A, B) Adenoviral expression of Pdi1 shRNA knocks down PDI1 levels in McA cells. (A) Relative abundance of mRNA expressed in control (NSi) and Pdi1-knockdown (PDI1i) cells. mRNA levels were normalized to actin levels. Values are relative to mRNA levels of control cells. *p < 0.01 vs. NSi. (B) PDI1 protein levels in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cell lysates were subjected to immunoblotting analyses using PDI1 and vinculin antibodies. (C–E) Pdi1-knockdown cells are more sensitive to ER stress. (C, D) Cell survival was measured by cell counting after treatment with different concentration of Tm or Tg. *p < 0.01 vs. NSi in the same treatment group. (E) Protein levels of ER stress markers in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cells were treated with 0.2 μM Tg for indicated time periods, and cell lysates were immunoblotted for cleaved caspase 3, PARP, eIF2α-P, CHOP, and BiP. (F) ER redox balance is not altered in Pdi1-knockdown cells. Cells expressing Grx-roGFP-iEER were treated with 10 mM DTT for 10 min and washed out. The oxidation status of Grx-roGFP-iEER was analyzed by nonreducing SDS–PAGE and immunoblotting with GFP, KDEL (GRP78 and GRP94), and PDI antibodies.
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Figure 1: Knockdown of Pdi1 sensitizes cells to ER stress. (A, B) Adenoviral expression of Pdi1 shRNA knocks down PDI1 levels in McA cells. (A) Relative abundance of mRNA expressed in control (NSi) and Pdi1-knockdown (PDI1i) cells. mRNA levels were normalized to actin levels. Values are relative to mRNA levels of control cells. *p < 0.01 vs. NSi. (B) PDI1 protein levels in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cell lysates were subjected to immunoblotting analyses using PDI1 and vinculin antibodies. (C–E) Pdi1-knockdown cells are more sensitive to ER stress. (C, D) Cell survival was measured by cell counting after treatment with different concentration of Tm or Tg. *p < 0.01 vs. NSi in the same treatment group. (E) Protein levels of ER stress markers in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cells were treated with 0.2 μM Tg for indicated time periods, and cell lysates were immunoblotted for cleaved caspase 3, PARP, eIF2α-P, CHOP, and BiP. (F) ER redox balance is not altered in Pdi1-knockdown cells. Cells expressing Grx-roGFP-iEER were treated with 10 mM DTT for 10 min and washed out. The oxidation status of Grx-roGFP-iEER was analyzed by nonreducing SDS–PAGE and immunoblotting with GFP, KDEL (GRP78 and GRP94), and PDI antibodies.

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.


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 sensitizes cells to ER stress. (A, B) Adenoviral expression of Pdi1 shRNA knocks down PDI1 levels in McA cells. (A) Relative abundance of mRNA expressed in control (NSi) and Pdi1-knockdown (PDI1i) cells. mRNA levels were normalized to actin levels. Values are relative to mRNA levels of control cells. *p < 0.01 vs. NSi. (B) PDI1 protein levels in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cell lysates were subjected to immunoblotting analyses using PDI1 and vinculin antibodies. (C–E) Pdi1-knockdown cells are more sensitive to ER stress. (C, D) Cell survival was measured by cell counting after treatment with different concentration of Tm or Tg. *p < 0.01 vs. NSi in the same treatment group. (E) Protein levels of ER stress markers in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cells were treated with 0.2 μM Tg for indicated time periods, and cell lysates were immunoblotted for cleaved caspase 3, PARP, eIF2α-P, CHOP, and BiP. (F) ER redox balance is not altered in Pdi1-knockdown cells. Cells expressing Grx-roGFP-iEER were treated with 10 mM DTT for 10 min and washed out. The oxidation status of Grx-roGFP-iEER was analyzed by nonreducing SDS–PAGE and immunoblotting with GFP, KDEL (GRP78 and GRP94), and PDI antibodies.
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Figure 1: Knockdown of Pdi1 sensitizes cells to ER stress. (A, B) Adenoviral expression of Pdi1 shRNA knocks down PDI1 levels in McA cells. (A) Relative abundance of mRNA expressed in control (NSi) and Pdi1-knockdown (PDI1i) cells. mRNA levels were normalized to actin levels. Values are relative to mRNA levels of control cells. *p < 0.01 vs. NSi. (B) PDI1 protein levels in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cell lysates were subjected to immunoblotting analyses using PDI1 and vinculin antibodies. (C–E) Pdi1-knockdown cells are more sensitive to ER stress. (C, D) Cell survival was measured by cell counting after treatment with different concentration of Tm or Tg. *p < 0.01 vs. NSi in the same treatment group. (E) Protein levels of ER stress markers in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cells were treated with 0.2 μM Tg for indicated time periods, and cell lysates were immunoblotted for cleaved caspase 3, PARP, eIF2α-P, CHOP, and BiP. (F) ER redox balance is not altered in Pdi1-knockdown cells. Cells expressing Grx-roGFP-iEER were treated with 10 mM DTT for 10 min and washed out. The oxidation status of Grx-roGFP-iEER was analyzed by nonreducing SDS–PAGE and immunoblotting with GFP, KDEL (GRP78 and GRP94), and PDI antibodies.
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.

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