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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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Effect of dNP expression on intracellular localization of components of the Ub-proteasome pathway. 143B cells infected for 4 h with VV-dNPpepGFP were incubated for an additional 4 h in the presence of 10 μM zLLL, fixed, permeabilized and stained for cellular components using the antibodies indicated. dNPpepGFP was located by its autofluorescence. Gray-scale images in each column are merged on the bottom with the color indicated by the text describing the antibody specificity. Arrows point to the MTOC. Bar, 10 μm.
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Figure 5: Effect of dNP expression on intracellular localization of components of the Ub-proteasome pathway. 143B cells infected for 4 h with VV-dNPpepGFP were incubated for an additional 4 h in the presence of 10 μM zLLL, fixed, permeabilized and stained for cellular components using the antibodies indicated. dNPpepGFP was located by its autofluorescence. Gray-scale images in each column are merged on the bottom with the color indicated by the text describing the antibody specificity. Arrows point to the MTOC. Bar, 10 μm.

Mentions: We colocalized the focal staining of dNPpep with antibodies specific for defined cellular structures. The nuclear structures colocalized with those stained by a mouse mAb (Fig. 3) or human autoimmune antibodies (see Fig. 5) specific for proteins present in PODs. PODs are enigmatic 0.3–1.0-μm-diameter macromolecular complexes comprised of >20 different proteins that are attached to the nuclear matrix (Sternsdorf et al. 1997). The cytoplasmic structure surrounded the staining obtained with a mAb specific for γ-tubulin, which identifies the pair of centrioles present at the microtubule organizing center (MTOC), the site where microtubules originate.


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)

Effect of dNP expression on intracellular localization of components of the Ub-proteasome pathway. 143B cells infected for 4 h with VV-dNPpepGFP were incubated for an additional 4 h in the presence of 10 μM zLLL, fixed, permeabilized and stained for cellular components using the antibodies indicated. dNPpepGFP was located by its autofluorescence. Gray-scale images in each column are merged on the bottom with the color indicated by the text describing the antibody specificity. Arrows point to the MTOC. Bar, 10 μm.
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Related In: Results  -  Collection

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

Figure 5: Effect of dNP expression on intracellular localization of components of the Ub-proteasome pathway. 143B cells infected for 4 h with VV-dNPpepGFP were incubated for an additional 4 h in the presence of 10 μM zLLL, fixed, permeabilized and stained for cellular components using the antibodies indicated. dNPpepGFP was located by its autofluorescence. Gray-scale images in each column are merged on the bottom with the color indicated by the text describing the antibody specificity. Arrows point to the MTOC. Bar, 10 μm.
Mentions: We colocalized the focal staining of dNPpep with antibodies specific for defined cellular structures. The nuclear structures colocalized with those stained by a mouse mAb (Fig. 3) or human autoimmune antibodies (see Fig. 5) specific for proteins present in PODs. PODs are enigmatic 0.3–1.0-μm-diameter macromolecular complexes comprised of >20 different proteins that are attached to the nuclear matrix (Sternsdorf et al. 1997). The cytoplasmic structure surrounded the staining obtained with a mAb specific for γ-tubulin, which identifies the pair of centrioles present at the microtubule organizing center (MTOC), the site where microtubules originate.

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