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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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Related in: MedlinePlus

Proteasome-mediated in situ degradation of dNPpep and generation of antigenic peptides. 143B cells infected for 4 h with VV-dNPpepGFP were incubated for an additional 90 min in the presence of 10 μM zLLL in the absence (−90′ zLLL) or presence of protein synthesis inhibitors emetine (25 μM) and cycloheximide (25 μM) (−90′ zLLL EC) and then fixed, or washed, and incubated for 4 h in protein synthesis inhibitors in the absence (240′ EC) or presence of zLLL (240′ zLLL EC) and then fixed. Cells were permeabilized and stained using PBC antiserum. dNPpepGFP was located by its autofluorescence. Bar, 10 μm.
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Figure 8: Proteasome-mediated in situ degradation of dNPpep and generation of antigenic peptides. 143B cells infected for 4 h with VV-dNPpepGFP were incubated for an additional 90 min in the presence of 10 μM zLLL in the absence (−90′ zLLL) or presence of protein synthesis inhibitors emetine (25 μM) and cycloheximide (25 μM) (−90′ zLLL EC) and then fixed, or washed, and incubated for 4 h in protein synthesis inhibitors in the absence (240′ EC) or presence of zLLL (240′ zLLL EC) and then fixed. Cells were permeabilized and stained using PBC antiserum. dNPpepGFP was located by its autofluorescence. Bar, 10 μm.

Mentions: The accumulation of dNPpep in PODs and the MTOC is not simply the result of prolonged overexpression. If cells were infected with rVV for 2–4 h in the absence of proteasome inhibitors, dNPpep was detected by anti-NH2 antibody staining in PODs and the MTOC as early as 30 min after adding zLLL in a small percentage of cells (data not shown), and by 90 min, in a high percentage of cells (see Fig. 8). In both circumstances, this represents the rapid accumulation of newly synthesized dNPpep, since it did not occur if protein synthesis inhibitors were added with zLLL.


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)

Proteasome-mediated in situ degradation of dNPpep and generation of antigenic peptides. 143B cells infected for 4 h with VV-dNPpepGFP were incubated for an additional 90 min in the presence of 10 μM zLLL in the absence (−90′ zLLL) or presence of protein synthesis inhibitors emetine (25 μM) and cycloheximide (25 μM) (−90′ zLLL EC) and then fixed, or washed, and incubated for 4 h in protein synthesis inhibitors in the absence (240′ EC) or presence of zLLL (240′ zLLL EC) and then fixed. Cells were permeabilized and stained using PBC antiserum. dNPpepGFP was located by its autofluorescence. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199725&req=5

Figure 8: Proteasome-mediated in situ degradation of dNPpep and generation of antigenic peptides. 143B cells infected for 4 h with VV-dNPpepGFP were incubated for an additional 90 min in the presence of 10 μM zLLL in the absence (−90′ zLLL) or presence of protein synthesis inhibitors emetine (25 μM) and cycloheximide (25 μM) (−90′ zLLL EC) and then fixed, or washed, and incubated for 4 h in protein synthesis inhibitors in the absence (240′ EC) or presence of zLLL (240′ zLLL EC) and then fixed. Cells were permeabilized and stained using PBC antiserum. dNPpepGFP was located by its autofluorescence. Bar, 10 μm.
Mentions: The accumulation of dNPpep in PODs and the MTOC is not simply the result of prolonged overexpression. If cells were infected with rVV for 2–4 h in the absence of proteasome inhibitors, dNPpep was detected by anti-NH2 antibody staining in PODs and the MTOC as early as 30 min after adding zLLL in a small percentage of cells (data not shown), and by 90 min, in a high percentage of cells (see Fig. 8). In both circumstances, this represents the rapid accumulation of newly synthesized dNPpep, since it did not occur if protein synthesis inhibitors were added with zLLL.

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