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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 proteasome inhibitors on the intracellular localization of components of the Ub-proteasome pathway in uninfected cells. 143B cells were incubated for 6 h in the presence of 21 μM zLLL, fixed, permeabilized, and stained for cellular components using the antibodies indicated. 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 6: Effect of proteasome inhibitors on the intracellular localization of components of the Ub-proteasome pathway in uninfected cells. 143B cells were incubated for 6 h in the presence of 21 μM zLLL, fixed, permeabilized, and stained for cellular components using the antibodies indicated. 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: In both live and fixed dNPpepGFP-expressing cells treated with zLLL for 4 h, fluorescent GFP was highly concentrated in PODs and the MTOC (Fig. 5, arrows point to the MTOC). The autofluorescence of dNPpepGFP indicates that the GFP domain is properly conformed, demonstrating that dNPpepGFP need not be completely denatured to localize to these structures. The accumulation of dNPpepGFP in these sites was accompanied by recruitment of poly Ub, proteasomes, and HSC70 from their normal diffuse distribution in the nucleus and cytoplasm, often to the extent that staining was reduced elsewhere in the cell (compare to Fig. 6; the distribution of proteasomes, not shown, is similar to poly Ub). While dNPpep and poly Ub filled the MTOC, in many cells proteasomes and HSC70 formed a ring around MTOC. The redistribution of cellular proteins is a specific effect of inhibiting proteasomes, as similar results were obtained with LC (data not shown). A survey of mAbs specific for other molecular chaperones (data not shown) revealed HSP27 recruited PODs similarly to HSC70 and somewhat less strongly to the MTOC, and HSP70 was recruited weakly to both sites. The distribution of a number of other cytosolic chaperones (HSP110, HSP90, HSP60, HSP56, HSP47, and HSP40) was not noticeably affected by dNPpep expression, and none were concentrated in either PODs or the MTOC.


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 proteasome inhibitors on the intracellular localization of components of the Ub-proteasome pathway in uninfected cells. 143B cells were incubated for 6 h in the presence of 21 μM zLLL, fixed, permeabilized, and stained for cellular components using the antibodies indicated. 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199725&req=5

Figure 6: Effect of proteasome inhibitors on the intracellular localization of components of the Ub-proteasome pathway in uninfected cells. 143B cells were incubated for 6 h in the presence of 21 μM zLLL, fixed, permeabilized, and stained for cellular components using the antibodies indicated. 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: In both live and fixed dNPpepGFP-expressing cells treated with zLLL for 4 h, fluorescent GFP was highly concentrated in PODs and the MTOC (Fig. 5, arrows point to the MTOC). The autofluorescence of dNPpepGFP indicates that the GFP domain is properly conformed, demonstrating that dNPpepGFP need not be completely denatured to localize to these structures. The accumulation of dNPpepGFP in these sites was accompanied by recruitment of poly Ub, proteasomes, and HSC70 from their normal diffuse distribution in the nucleus and cytoplasm, often to the extent that staining was reduced elsewhere in the cell (compare to Fig. 6; the distribution of proteasomes, not shown, is similar to poly Ub). While dNPpep and poly Ub filled the MTOC, in many cells proteasomes and HSC70 formed a ring around MTOC. The redistribution of cellular proteins is a specific effect of inhibiting proteasomes, as similar results were obtained with LC (data not shown). A survey of mAbs specific for other molecular chaperones (data not shown) revealed HSP27 recruited PODs similarly to HSC70 and somewhat less strongly to the MTOC, and HSP70 was recruited weakly to both sites. The distribution of a number of other cytosolic chaperones (HSP110, HSP90, HSP60, HSP56, HSP47, and HSP40) was not noticeably affected by dNPpep expression, and none were concentrated in either PODs or the MTOC.

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