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Intracellular localization of proteasomal degradation of a viral antigen.

Antón LC, Schubert U, Bacík I, Princiotta MF, Wearsch PA, Gibbs J, Day PM, Realini C, Rechsteiner MC, Bennink JR, Yewdell JW - J. Cell Biol. (1999)

Bottom Line: In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC).Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP.These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.

ABSTRACT
To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.

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dNPpep rescued by zLLL localizes in PODs and the MTOC. rVV-infected 143B cells were incubated for 3 h before the addition of 20 μM zLLL. After 6.5 h, cells were fixed, permeabilized, stained with the indicated antibodies, and imaged with the LCSM. The second row displays cells expressing NPpep, in the first and third, cells expressing dNPpep. In the bottom row, dNPpep expressing cells were first extracted with 1% NP-40 and then fixed with methanol/acetone (80:20) for 15 min at −20°C to enable staining with the γ-tubulin–specific mAb. Arrows point to the MTOC. Gray-scale images on the sides are merged in the middle with the color indicated by the text describing the antibody specificity. Bar, 10 μM.
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Figure 3: dNPpep rescued by zLLL localizes in PODs and the MTOC. rVV-infected 143B cells were incubated for 3 h before the addition of 20 μM zLLL. After 6.5 h, cells were fixed, permeabilized, stained with the indicated antibodies, and imaged with the LCSM. The second row displays cells expressing NPpep, in the first and third, cells expressing dNPpep. In the bottom row, dNPpep expressing cells were first extracted with 1% NP-40 and then fixed with methanol/acetone (80:20) for 15 min at −20°C to enable staining with the γ-tubulin–specific mAb. Arrows point to the MTOC. Gray-scale images on the sides are merged in the middle with the color indicated by the text describing the antibody specificity. Bar, 10 μM.

Mentions: This prediction was first confirmed by immunofluorescence of fixed and permeabilized rVV-infected cells using rabbit antibodies raised to the NH2 terminus of unmodified NP (anti-NH2). In the absence of proteasome inhibitors, staining of dNPpep observed using a laser scanning confocal microscope (LCSM) was only slightly above background autofluorescence levels (data not shown). In the presence of either LC (data not shown) or the reversible proteasome inhibitor zLLL, dNPpep was detected in three locations: weak staining of the nuclear body (excluding the nucleoli), and strong staining of small nuclear substructures (Fig. 3, first row) and, in some cells, a cytoplasmic structure that was often juxtanuclear (Fig. 3, third and fourth rows, arrows). This differs markedly from the staining pattern of NPpep (Fig. 3, second row), which like wild-type NP (not shown) strongly stains the nuclear body in the presence or absence (not shown) of proteasome inhibitors.


Intracellular localization of proteasomal degradation of a viral antigen.

Antón LC, Schubert U, Bacík I, Princiotta MF, Wearsch PA, Gibbs J, Day PM, Realini C, Rechsteiner MC, Bennink JR, Yewdell JW - J. Cell Biol. (1999)

dNPpep rescued by zLLL localizes in PODs and the MTOC. rVV-infected 143B cells were incubated for 3 h before the addition of 20 μM zLLL. After 6.5 h, cells were fixed, permeabilized, stained with the indicated antibodies, and imaged with the LCSM. The second row displays cells expressing NPpep, in the first and third, cells expressing dNPpep. In the bottom row, dNPpep expressing cells were first extracted with 1% NP-40 and then fixed with methanol/acetone (80:20) for 15 min at −20°C to enable staining with the γ-tubulin–specific mAb. Arrows point to the MTOC. Gray-scale images on the sides are merged in the middle with the color indicated by the text describing the antibody specificity. Bar, 10 μM.
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Related In: Results  -  Collection

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Figure 3: dNPpep rescued by zLLL localizes in PODs and the MTOC. rVV-infected 143B cells were incubated for 3 h before the addition of 20 μM zLLL. After 6.5 h, cells were fixed, permeabilized, stained with the indicated antibodies, and imaged with the LCSM. The second row displays cells expressing NPpep, in the first and third, cells expressing dNPpep. In the bottom row, dNPpep expressing cells were first extracted with 1% NP-40 and then fixed with methanol/acetone (80:20) for 15 min at −20°C to enable staining with the γ-tubulin–specific mAb. Arrows point to the MTOC. Gray-scale images on the sides are merged in the middle with the color indicated by the text describing the antibody specificity. Bar, 10 μM.
Mentions: This prediction was first confirmed by immunofluorescence of fixed and permeabilized rVV-infected cells using rabbit antibodies raised to the NH2 terminus of unmodified NP (anti-NH2). In the absence of proteasome inhibitors, staining of dNPpep observed using a laser scanning confocal microscope (LCSM) was only slightly above background autofluorescence levels (data not shown). In the presence of either LC (data not shown) or the reversible proteasome inhibitor zLLL, dNPpep was detected in three locations: weak staining of the nuclear body (excluding the nucleoli), and strong staining of small nuclear substructures (Fig. 3, first row) and, in some cells, a cytoplasmic structure that was often juxtanuclear (Fig. 3, third and fourth rows, arrows). This differs markedly from the staining pattern of NPpep (Fig. 3, second row), which like wild-type NP (not shown) strongly stains the nuclear body in the presence or absence (not shown) of proteasome inhibitors.

Bottom Line: In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC).Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP.These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.

ABSTRACT
To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.

Show MeSH
Related in: MedlinePlus