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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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Antigenic peptides are generated from a pool of dNPpep accumulated in the presence of proteasome inhibitor. 143B cells were coinfected with a rVV expressing Kb and mouse β2m and a rVV expressing the protein indicated. Cells were infected for 4 h in the presence of 10 μM zLLL to prevent the generation of Kb–Ova257-264 complexes, washed, and incubated for 4 h in protein synthesis inhibitors in the presence (bottom left histogram) or absence (bottom right histogram) of zLLL. As controls, cells were incubated for 8 h in the absence of inhibitors (top left histogram) or in the presence of protein synthesis inhibitors (top right histogram). Cells were indirectly stained with the 25-D1.16 mAb to quantitate the number of cell surface Kb-Ova257-264 complexes.
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Figure 9: Antigenic peptides are generated from a pool of dNPpep accumulated in the presence of proteasome inhibitor. 143B cells were coinfected with a rVV expressing Kb and mouse β2m and a rVV expressing the protein indicated. Cells were infected for 4 h in the presence of 10 μM zLLL to prevent the generation of Kb–Ova257-264 complexes, washed, and incubated for 4 h in protein synthesis inhibitors in the presence (bottom left histogram) or absence (bottom right histogram) of zLLL. As controls, cells were incubated for 8 h in the absence of inhibitors (top left histogram) or in the presence of protein synthesis inhibitors (top right histogram). Cells were indirectly stained with the 25-D1.16 mAb to quantitate the number of cell surface Kb-Ova257-264 complexes.

Mentions: We next related these findings to antigen processing. Kb was expressed in TK− cells by coinfection with a rVV expressing Kb and mouse β2-microglobulin, and the expression of cell surface Kb–Ova257-264 complexes quantitated cytofluorographically after indirect staining with the 25-D1.16 mAb (Fig. 9). Levels of background staining were controlled for by infection with VV-NP. As with L-Kb cells (Fig. 1), Kb–Ova257-264 complexes are produced more efficiently from dNPpep than NPpep; in this case the difference is even more pronounced (six- versus threefold increase in mean fluorescence). To correlate the disappearance of dNPpep from PODs and the MTOC with proteasome mediated generation of Ova257-264, cells were infected for 4 h in the continuous presence of zLLL, washed, and incubated for 4 h in the presence of protein synthesis inhibitors without (EC) or with zLLL (zLLL EC). In the continued presence of zLLL (zLLL EC) no complexes were generated from dNPpep or NPpep, since levels of staining of VV-NP–, VV-NPpep–, and VV-dNPpep–infected cells were identical. As described above (Fig. 1), zLLL had little effect on the generation of complexes by cells expressing the cytosolic minigene product, OvaM257-264. Removal of zLLL in the presence of protein synthesis inhibitors was accompanied by the generation of a signal in dNPpep-expressing cells above the staining of NP-expressing cells (EC). Although the shift in the curve is relatively small, it represents 31% of the signal obtained in the continuous absence of inhibitors (no inhibitor), and in absolute terms, roughly 1,000 Kb–Ova257-264 complexes, which is more than sufficient for triggering most T cells. The effectiveness of the protein synthesis inhibitors is clearly demonstrated by the background staining of cells treated with inhibitors from the initiation of the infection (EC start), even if cells were infected with the rVV expressing the cytosolic minigene product. In contrast to results with dNPpep, Kb–Ova257-264 complexes were not generated from NPpep upon removal of zLLL. These findings indicate that removal of zLLL from cells allows proteasomes to generate peptides from the dNPpep that accumulates in the cells (but not from NPpep), and is consistent with the idea the peptides (or their precursors) are generated at PODs, the MTOC, or at both of these locations.


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)

Antigenic peptides are generated from a pool of dNPpep accumulated in the presence of proteasome inhibitor. 143B cells were coinfected with a rVV expressing Kb and mouse β2m and a rVV expressing the protein indicated. Cells were infected for 4 h in the presence of 10 μM zLLL to prevent the generation of Kb–Ova257-264 complexes, washed, and incubated for 4 h in protein synthesis inhibitors in the presence (bottom left histogram) or absence (bottom right histogram) of zLLL. As controls, cells were incubated for 8 h in the absence of inhibitors (top left histogram) or in the presence of protein synthesis inhibitors (top right histogram). Cells were indirectly stained with the 25-D1.16 mAb to quantitate the number of cell surface Kb-Ova257-264 complexes.
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Related In: Results  -  Collection

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Figure 9: Antigenic peptides are generated from a pool of dNPpep accumulated in the presence of proteasome inhibitor. 143B cells were coinfected with a rVV expressing Kb and mouse β2m and a rVV expressing the protein indicated. Cells were infected for 4 h in the presence of 10 μM zLLL to prevent the generation of Kb–Ova257-264 complexes, washed, and incubated for 4 h in protein synthesis inhibitors in the presence (bottom left histogram) or absence (bottom right histogram) of zLLL. As controls, cells were incubated for 8 h in the absence of inhibitors (top left histogram) or in the presence of protein synthesis inhibitors (top right histogram). Cells were indirectly stained with the 25-D1.16 mAb to quantitate the number of cell surface Kb-Ova257-264 complexes.
Mentions: We next related these findings to antigen processing. Kb was expressed in TK− cells by coinfection with a rVV expressing Kb and mouse β2-microglobulin, and the expression of cell surface Kb–Ova257-264 complexes quantitated cytofluorographically after indirect staining with the 25-D1.16 mAb (Fig. 9). Levels of background staining were controlled for by infection with VV-NP. As with L-Kb cells (Fig. 1), Kb–Ova257-264 complexes are produced more efficiently from dNPpep than NPpep; in this case the difference is even more pronounced (six- versus threefold increase in mean fluorescence). To correlate the disappearance of dNPpep from PODs and the MTOC with proteasome mediated generation of Ova257-264, cells were infected for 4 h in the continuous presence of zLLL, washed, and incubated for 4 h in the presence of protein synthesis inhibitors without (EC) or with zLLL (zLLL EC). In the continued presence of zLLL (zLLL EC) no complexes were generated from dNPpep or NPpep, since levels of staining of VV-NP–, VV-NPpep–, and VV-dNPpep–infected cells were identical. As described above (Fig. 1), zLLL had little effect on the generation of complexes by cells expressing the cytosolic minigene product, OvaM257-264. Removal of zLLL in the presence of protein synthesis inhibitors was accompanied by the generation of a signal in dNPpep-expressing cells above the staining of NP-expressing cells (EC). Although the shift in the curve is relatively small, it represents 31% of the signal obtained in the continuous absence of inhibitors (no inhibitor), and in absolute terms, roughly 1,000 Kb–Ova257-264 complexes, which is more than sufficient for triggering most T cells. The effectiveness of the protein synthesis inhibitors is clearly demonstrated by the background staining of cells treated with inhibitors from the initiation of the infection (EC start), even if cells were infected with the rVV expressing the cytosolic minigene product. In contrast to results with dNPpep, Kb–Ova257-264 complexes were not generated from NPpep upon removal of zLLL. These findings indicate that removal of zLLL from cells allows proteasomes to generate peptides from the dNPpep that accumulates in the cells (but not from NPpep), and is consistent with the idea the peptides (or their precursors) are generated at PODs, the MTOC, or at both of these locations.

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