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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 apoB synthesis and secretion in McA cells. (A, B) Pdi1 knockdown in McA cells reduces apoB100 protein but not mRNA levels. (A) Relative abundance of apoB and MTP mRNAs in control (NSi) and Pdi1-knockdown (PDI1i) cells. Relative abundance was normalized to actin mRNA levels. Values are relative to mRNA levels of control cells. *p < 0.01 vs. NSi (B) ApoB100 and MTP protein levels in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cell lysates were immunoblotted for PDI1, apoB100, MTP, and actin. (C) Pdi1-knockdown (PDI1i) in McA cells reduces apoB100 synthesis and secretion. Pulse-chase analysis of intracellular and secreted apoB100 was performed in control (NSi) and Pdi1-knockdown (PDI1i) McA cells. Double asterisks represent a nonspecific band. (D) Proteasomal degradation reduces apoB100 levels in McA cells. Pulse-chase analysis of intracellular apoB100 and secreted apoB was performed in control (NSi) and Pdi1-knockdown (PDI1i) McA cells in the absence (control) or presence of 10 μM MG132. (E) The amount of apoB100 in intracellular and secreted fractions was quantified by ImageJ. Recovery rates of apoB100 are presented as percentage of the sum of apoB100 (intracellular plus secreted) over total apoB100 at zero time point of pulse-chase experiments. Pulse-chase experiments were repeated at least twice.
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Figure 2: Knockdown of Pdi1 decreases apoB synthesis and secretion in McA cells. (A, B) Pdi1 knockdown in McA cells reduces apoB100 protein but not mRNA levels. (A) Relative abundance of apoB and MTP mRNAs in control (NSi) and Pdi1-knockdown (PDI1i) cells. Relative abundance was normalized to actin mRNA levels. Values are relative to mRNA levels of control cells. *p < 0.01 vs. NSi (B) ApoB100 and MTP protein levels in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cell lysates were immunoblotted for PDI1, apoB100, MTP, and actin. (C) Pdi1-knockdown (PDI1i) in McA cells reduces apoB100 synthesis and secretion. Pulse-chase analysis of intracellular and secreted apoB100 was performed in control (NSi) and Pdi1-knockdown (PDI1i) McA cells. Double asterisks represent a nonspecific band. (D) Proteasomal degradation reduces apoB100 levels in McA cells. Pulse-chase analysis of intracellular apoB100 and secreted apoB was performed in control (NSi) and Pdi1-knockdown (PDI1i) McA cells in the absence (control) or presence of 10 μM MG132. (E) The amount of apoB100 in intracellular and secreted fractions was quantified by ImageJ. Recovery rates of apoB100 are presented as percentage of the sum of apoB100 (intracellular plus secreted) over total apoB100 at zero time point of pulse-chase experiments. Pulse-chase experiments were repeated at least twice.

Mentions: We next examined protein levels of apoB100 and MTP and found that Pdi1 knockdown drastically decreased intracellular apoB100 levels by ∼50% without altering its mRNA expression. Pdi1 knockdown had a relatively milder effect on MTP, with a ∼20% decrease in steady-state protein levels (Figure 2, A and B). Nonetheless, these observations indicate that PDI1 plays an essential role in maintaining intracellular apoB100 levels.


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 apoB synthesis and secretion in McA cells. (A, B) Pdi1 knockdown in McA cells reduces apoB100 protein but not mRNA levels. (A) Relative abundance of apoB and MTP mRNAs in control (NSi) and Pdi1-knockdown (PDI1i) cells. Relative abundance was normalized to actin mRNA levels. Values are relative to mRNA levels of control cells. *p < 0.01 vs. NSi (B) ApoB100 and MTP protein levels in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cell lysates were immunoblotted for PDI1, apoB100, MTP, and actin. (C) Pdi1-knockdown (PDI1i) in McA cells reduces apoB100 synthesis and secretion. Pulse-chase analysis of intracellular and secreted apoB100 was performed in control (NSi) and Pdi1-knockdown (PDI1i) McA cells. Double asterisks represent a nonspecific band. (D) Proteasomal degradation reduces apoB100 levels in McA cells. Pulse-chase analysis of intracellular apoB100 and secreted apoB was performed in control (NSi) and Pdi1-knockdown (PDI1i) McA cells in the absence (control) or presence of 10 μM MG132. (E) The amount of apoB100 in intracellular and secreted fractions was quantified by ImageJ. Recovery rates of apoB100 are presented as percentage of the sum of apoB100 (intracellular plus secreted) over total apoB100 at zero time point of pulse-chase experiments. Pulse-chase experiments were repeated at least twice.
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Figure 2: Knockdown of Pdi1 decreases apoB synthesis and secretion in McA cells. (A, B) Pdi1 knockdown in McA cells reduces apoB100 protein but not mRNA levels. (A) Relative abundance of apoB and MTP mRNAs in control (NSi) and Pdi1-knockdown (PDI1i) cells. Relative abundance was normalized to actin mRNA levels. Values are relative to mRNA levels of control cells. *p < 0.01 vs. NSi (B) ApoB100 and MTP protein levels in control (NSi) and Pdi1-knockdown (PDI1i) cells. Cell lysates were immunoblotted for PDI1, apoB100, MTP, and actin. (C) Pdi1-knockdown (PDI1i) in McA cells reduces apoB100 synthesis and secretion. Pulse-chase analysis of intracellular and secreted apoB100 was performed in control (NSi) and Pdi1-knockdown (PDI1i) McA cells. Double asterisks represent a nonspecific band. (D) Proteasomal degradation reduces apoB100 levels in McA cells. Pulse-chase analysis of intracellular apoB100 and secreted apoB was performed in control (NSi) and Pdi1-knockdown (PDI1i) McA cells in the absence (control) or presence of 10 μM MG132. (E) The amount of apoB100 in intracellular and secreted fractions was quantified by ImageJ. Recovery rates of apoB100 are presented as percentage of the sum of apoB100 (intracellular plus secreted) over total apoB100 at zero time point of pulse-chase experiments. Pulse-chase experiments were repeated at least twice.
Mentions: We next examined protein levels of apoB100 and MTP and found that Pdi1 knockdown drastically decreased intracellular apoB100 levels by ∼50% without altering its mRNA expression. Pdi1 knockdown had a relatively milder effect on MTP, with a ∼20% decrease in steady-state protein levels (Figure 2, A and B). Nonetheless, these observations indicate that PDI1 plays an essential role in maintaining intracellular apoB100 levels.

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