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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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Reduced apoB100 oxidative folding decreases apoB lipidation. (A) PDI1 forms intermolecular disulfide bond(s) with apoB100. Lysates from cells that stably express HA-tagged wild-type (Pw: HA-PDI1) or substrate-trap mutant (Pc: HA-PDICXXA) of human PDI1 were immunoprecipitated with apoB100 antibody and analyzed by nonreducing SDS–PAGE and immunoblotting against HA antibody. The circle indicates PDI1 complexed with apoB100. IP, immuno­precipitated protein complex; T, total cell lysates. (B) Impaired apoB100 oxidative folding decreases apoB100 lipidation. Secreted apoB100-containing lipoproteins from McA cells were separated by DGUC as described in Figure 3 and analyzed by nonreducing gel electrophoresis. (C) 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.
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Figure 5: Reduced apoB100 oxidative folding decreases apoB lipidation. (A) PDI1 forms intermolecular disulfide bond(s) with apoB100. Lysates from cells that stably express HA-tagged wild-type (Pw: HA-PDI1) or substrate-trap mutant (Pc: HA-PDICXXA) of human PDI1 were immunoprecipitated with apoB100 antibody and analyzed by nonreducing SDS–PAGE and immunoblotting against HA antibody. The circle indicates PDI1 complexed with apoB100. IP, immuno­precipitated protein complex; T, total cell lysates. (B) Impaired apoB100 oxidative folding decreases apoB100 lipidation. Secreted apoB100-containing lipoproteins from McA cells were separated by DGUC as described in Figure 3 and analyzed by nonreducing gel electrophoresis. (C) 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.

Mentions: To confirm that apoB100 is a substrate for PDI1, we analyzed the apoB100 protein complex in Pdi1-knockdown McA cells that stably express either HA-PDI1 or HA-PDI1CXXA as described before. Cell lysates were immunoprecipitated using apoB antibody, and the immunocomplexes were analyzed by SDS–PAGE under nonreducing conditions, followed by immunoblotting with anti-HA antibody. If apoB100 is a substrate of PDI1, the substrate-trap mutant of PDI1 should be present in covalent association with apoB100 under nonreducing conditions. Indeed, we detected HA-PDI1CXXA in the apoB100 immunocomplex under nonreducing conditions (Figure 5A), indicating that the cysteines of the CXXA motif in substrate-trap mutant PDI1 can form disulfide bonds with cysteine residues of apoB100. This observation indicates that PDI1 promotes formation of disulfide bonds in apoB100 via intermolecular disulfide bonds. Thus PDI1 is important for stabilizing apoB100 structure by serving as a thiol-disulfide oxidoreductase.


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)

Reduced apoB100 oxidative folding decreases apoB lipidation. (A) PDI1 forms intermolecular disulfide bond(s) with apoB100. Lysates from cells that stably express HA-tagged wild-type (Pw: HA-PDI1) or substrate-trap mutant (Pc: HA-PDICXXA) of human PDI1 were immunoprecipitated with apoB100 antibody and analyzed by nonreducing SDS–PAGE and immunoblotting against HA antibody. The circle indicates PDI1 complexed with apoB100. IP, immuno­precipitated protein complex; T, total cell lysates. (B) Impaired apoB100 oxidative folding decreases apoB100 lipidation. Secreted apoB100-containing lipoproteins from McA cells were separated by DGUC as described in Figure 3 and analyzed by nonreducing gel electrophoresis. (C) 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.
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Related In: Results  -  Collection

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Figure 5: Reduced apoB100 oxidative folding decreases apoB lipidation. (A) PDI1 forms intermolecular disulfide bond(s) with apoB100. Lysates from cells that stably express HA-tagged wild-type (Pw: HA-PDI1) or substrate-trap mutant (Pc: HA-PDICXXA) of human PDI1 were immunoprecipitated with apoB100 antibody and analyzed by nonreducing SDS–PAGE and immunoblotting against HA antibody. The circle indicates PDI1 complexed with apoB100. IP, immuno­precipitated protein complex; T, total cell lysates. (B) Impaired apoB100 oxidative folding decreases apoB100 lipidation. Secreted apoB100-containing lipoproteins from McA cells were separated by DGUC as described in Figure 3 and analyzed by nonreducing gel electrophoresis. (C) 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.
Mentions: To confirm that apoB100 is a substrate for PDI1, we analyzed the apoB100 protein complex in Pdi1-knockdown McA cells that stably express either HA-PDI1 or HA-PDI1CXXA as described before. Cell lysates were immunoprecipitated using apoB antibody, and the immunocomplexes were analyzed by SDS–PAGE under nonreducing conditions, followed by immunoblotting with anti-HA antibody. If apoB100 is a substrate of PDI1, the substrate-trap mutant of PDI1 should be present in covalent association with apoB100 under nonreducing conditions. Indeed, we detected HA-PDI1CXXA in the apoB100 immunocomplex under nonreducing conditions (Figure 5A), indicating that the cysteines of the CXXA motif in substrate-trap mutant PDI1 can form disulfide bonds with cysteine residues of apoB100. This observation indicates that PDI1 promotes formation of disulfide bonds in apoB100 via intermolecular disulfide bonds. Thus PDI1 is important for stabilizing apoB100 structure by serving as a thiol-disulfide oxidoreductase.

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