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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 oxidative folding in McA cells. (A) Pdi1 knockdown decreases oxidative folding of apoB100. Pulse-chase analysis of intracellular and secreted apoB100 in control (NSi) and Pdi1-knockdown (PDI1i) McA cells by nonreducing gel electrophoresis. The bracket indicates both oxidized and reduced form of apoB100 in nonreducing gel. (B) Pdi1-knockdown selectively reduces oxidative folding of apoB100. Oxidative folding of intracellular apoB100, albumin, and transferrin were measured by reducing (first two lanes) and nonreducing gel electrophoresis. Ox, oxidized form, and Re, reduced form, of the protein. (C) Oxidative folding of apoB100 requires catalytically active PDI1. Oxidative folding of intracellular apoB100, albumin, and transferrin was measured by nonreducing gel electrophoresis in control (NSi) cells, Pdi1-knockdown (PDI1i) and Pdi1-knockdown (PDI1i) cells with exogenous expression of HA-tagged substrate-trap mutant (huPDImu) or wild-type (huPDI) human PDI1. (D) Oxidative folding of apoB100 does not require MTP activity. Oxidative folding of intracellular apoB100 and albumin were analyzed by nonreducing gel electrophoresis in the absence or presence of an MTP inhibitor (CP-10447) at 0, 0.3, or 3 μM.
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Figure 4: Knockdown of Pdi1 decreases apoB100 oxidative folding in McA cells. (A) Pdi1 knockdown decreases oxidative folding of apoB100. Pulse-chase analysis of intracellular and secreted apoB100 in control (NSi) and Pdi1-knockdown (PDI1i) McA cells by nonreducing gel electrophoresis. The bracket indicates both oxidized and reduced form of apoB100 in nonreducing gel. (B) Pdi1-knockdown selectively reduces oxidative folding of apoB100. Oxidative folding of intracellular apoB100, albumin, and transferrin were measured by reducing (first two lanes) and nonreducing gel electrophoresis. Ox, oxidized form, and Re, reduced form, of the protein. (C) Oxidative folding of apoB100 requires catalytically active PDI1. Oxidative folding of intracellular apoB100, albumin, and transferrin was measured by nonreducing gel electrophoresis in control (NSi) cells, Pdi1-knockdown (PDI1i) and Pdi1-knockdown (PDI1i) cells with exogenous expression of HA-tagged substrate-trap mutant (huPDImu) or wild-type (huPDI) human PDI1. (D) Oxidative folding of apoB100 does not require MTP activity. Oxidative folding of intracellular apoB100 and albumin were analyzed by nonreducing gel electrophoresis in the absence or presence of an MTP inhibitor (CP-10447) at 0, 0.3, or 3 μM.

Mentions: Previous work suggested that the oxidoreductase activity of PDI1 is required for MTP-independent steps of apoB100 synthesis and secretion (Wang et al., 1997). To explore this possibility in more detail, we used pulse-chase experiments in which newly synthesized proteins were resolved by nonreducing gel electrophoresis to allow us to test whether Pdi1 knockdown modifies apoB disulfide bond formation. We found that intracellular and secreted apoB100 migrated more slowly at the later chase time points in Pdi1-knockdown cells, raising the possibility that disulfide bond formation is delayed for newly synthesized apoB100 (Figure 4A, asterisk). To our surprise, apoB100 with partially oxidized disulfide bonds were also secreted in Pdi1-knockdown cells, suggesting that these apoB100 molecules undergo sufficient lipidation to attenuate posttranslational degradation.


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 oxidative folding in McA cells. (A) Pdi1 knockdown decreases oxidative folding of apoB100. Pulse-chase analysis of intracellular and secreted apoB100 in control (NSi) and Pdi1-knockdown (PDI1i) McA cells by nonreducing gel electrophoresis. The bracket indicates both oxidized and reduced form of apoB100 in nonreducing gel. (B) Pdi1-knockdown selectively reduces oxidative folding of apoB100. Oxidative folding of intracellular apoB100, albumin, and transferrin were measured by reducing (first two lanes) and nonreducing gel electrophoresis. Ox, oxidized form, and Re, reduced form, of the protein. (C) Oxidative folding of apoB100 requires catalytically active PDI1. Oxidative folding of intracellular apoB100, albumin, and transferrin was measured by nonreducing gel electrophoresis in control (NSi) cells, Pdi1-knockdown (PDI1i) and Pdi1-knockdown (PDI1i) cells with exogenous expression of HA-tagged substrate-trap mutant (huPDImu) or wild-type (huPDI) human PDI1. (D) Oxidative folding of apoB100 does not require MTP activity. Oxidative folding of intracellular apoB100 and albumin were analyzed by nonreducing gel electrophoresis in the absence or presence of an MTP inhibitor (CP-10447) at 0, 0.3, or 3 μM.
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Figure 4: Knockdown of Pdi1 decreases apoB100 oxidative folding in McA cells. (A) Pdi1 knockdown decreases oxidative folding of apoB100. Pulse-chase analysis of intracellular and secreted apoB100 in control (NSi) and Pdi1-knockdown (PDI1i) McA cells by nonreducing gel electrophoresis. The bracket indicates both oxidized and reduced form of apoB100 in nonreducing gel. (B) Pdi1-knockdown selectively reduces oxidative folding of apoB100. Oxidative folding of intracellular apoB100, albumin, and transferrin were measured by reducing (first two lanes) and nonreducing gel electrophoresis. Ox, oxidized form, and Re, reduced form, of the protein. (C) Oxidative folding of apoB100 requires catalytically active PDI1. Oxidative folding of intracellular apoB100, albumin, and transferrin was measured by nonreducing gel electrophoresis in control (NSi) cells, Pdi1-knockdown (PDI1i) and Pdi1-knockdown (PDI1i) cells with exogenous expression of HA-tagged substrate-trap mutant (huPDImu) or wild-type (huPDI) human PDI1. (D) Oxidative folding of apoB100 does not require MTP activity. Oxidative folding of intracellular apoB100 and albumin were analyzed by nonreducing gel electrophoresis in the absence or presence of an MTP inhibitor (CP-10447) at 0, 0.3, or 3 μM.
Mentions: Previous work suggested that the oxidoreductase activity of PDI1 is required for MTP-independent steps of apoB100 synthesis and secretion (Wang et al., 1997). To explore this possibility in more detail, we used pulse-chase experiments in which newly synthesized proteins were resolved by nonreducing gel electrophoresis to allow us to test whether Pdi1 knockdown modifies apoB disulfide bond formation. We found that intracellular and secreted apoB100 migrated more slowly at the later chase time points in Pdi1-knockdown cells, raising the possibility that disulfide bond formation is delayed for newly synthesized apoB100 (Figure 4A, asterisk). To our surprise, apoB100 with partially oxidized disulfide bonds were also secreted in Pdi1-knockdown cells, suggesting that these apoB100 molecules undergo sufficient lipidation to attenuate posttranslational degradation.

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