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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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Proteasome-dependent production of Ova257-264 from VV-encoded proteins. L-Kb cells incubated for 90 min in the absence (top) or presence (bottom) of 50 μM LC were infected for 8 h with the indicated rVV in the presence or absence of LC, respectively. Cells were stained with 25-D1.16 mAb and analyzed by cytofluorography.
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Figure 1: Proteasome-dependent production of Ova257-264 from VV-encoded proteins. L-Kb cells incubated for 90 min in the absence (top) or presence (bottom) of 50 μM LC were infected for 8 h with the indicated rVV in the presence or absence of LC, respectively. Cells were stained with 25-D1.16 mAb and analyzed by cytofluorography.

Mentions: After 6 h of infection of L-Kb cells with rVVs expressing NPpep or dNPpep, approximately threefold more Kb–Ova257-264 complexes were present on the surface of VV-dNPpep–infected cells as determined cytofluorographically after indirect immunofluorescence (Fig. 1, top histogram). Incubation of cells with the highly specific irreversible proteasome inhibitor LC resulted in the nearly complete inhibition of complex expression from the chimeric proteins and from OVA, the parent protein (Fig. 1, bottom histogram). There was only a slight effect on cells infected with a rVV expressing Ova257-264 as a cytosolic minigene product (a single Met is appended to the NH2 terminus to enable efficient translation), consistent with the interpretation that LC acts by preventing proteasome liberation of Ova257-264 (or a proteolytic intermediate) from NPpep, dNPpep, and OVA, and not by interfering with VV gene expression or delivery and loading of peptides onto Kb molecules.


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-dependent production of Ova257-264 from VV-encoded proteins. L-Kb cells incubated for 90 min in the absence (top) or presence (bottom) of 50 μM LC were infected for 8 h with the indicated rVV in the presence or absence of LC, respectively. Cells were stained with 25-D1.16 mAb and analyzed by cytofluorography.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Proteasome-dependent production of Ova257-264 from VV-encoded proteins. L-Kb cells incubated for 90 min in the absence (top) or presence (bottom) of 50 μM LC were infected for 8 h with the indicated rVV in the presence or absence of LC, respectively. Cells were stained with 25-D1.16 mAb and analyzed by cytofluorography.
Mentions: After 6 h of infection of L-Kb cells with rVVs expressing NPpep or dNPpep, approximately threefold more Kb–Ova257-264 complexes were present on the surface of VV-dNPpep–infected cells as determined cytofluorographically after indirect immunofluorescence (Fig. 1, top histogram). Incubation of cells with the highly specific irreversible proteasome inhibitor LC resulted in the nearly complete inhibition of complex expression from the chimeric proteins and from OVA, the parent protein (Fig. 1, bottom histogram). There was only a slight effect on cells infected with a rVV expressing Ova257-264 as a cytosolic minigene product (a single Met is appended to the NH2 terminus to enable efficient translation), consistent with the interpretation that LC acts by preventing proteasome liberation of Ova257-264 (or a proteolytic intermediate) from NPpep, dNPpep, and OVA, and not by interfering with VV gene expression or delivery and loading of peptides onto Kb molecules.

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