Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of canavanine on intracellular localization of NP and cellular proteins. 143B cells infected for 2 h with VV-NP were incubated for an additional 6 h in the presence of 15 mM canavanine. Cells were fixed, permeabilized, and stained with the antibodies indicated. NP stained by anti-COOH antisera is shown in the right column. Gray-scale images on the left and right are merged in the middle column 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


getmorefigures.php?uid=PMC2199725&req=5

Figure 7: Effect of canavanine on intracellular localization of NP and cellular proteins. 143B cells infected for 2 h with VV-NP were incubated for an additional 6 h in the presence of 15 mM canavanine. Cells were fixed, permeabilized, and stained with the antibodies indicated. NP stained by anti-COOH antisera is shown in the right column. Gray-scale images on the left and right are merged in the middle column with the color indicated by the text describing the antibody specificity. Arrows point to the MTOC. Bar, 10 μm.

Mentions: It was even possible to induce wild-type NP to localize to PODs and the MTOC by exposing VV-NP–infected to cells to canavanine, an amino acid analogue of Arg that induces protein misfolding (Fig. 7). NP in PODs and the MTOC was detected by anti-COOH but not anti-NH2 Abs, possibly due to the replacement of Arg in the NH2-terminal peptide with canavanine (the COOH peptide used for Ab generation does not contain Arg). In addition, large amounts of NP were now detected in the cytosol, an effect possibly related to canavanine modification of the nuclear localization signal. NP in PODs and MTOC recruited poly Ub, proteasomes, and HSC70. Notably this occurred in the absence of proteasome inhibitors, suggesting that the degradation machinery (which could also be affected by canavanine) was compromised under these conditions, either as a result of having to cope with vast quantities of proteins misfolded by incorporation of canavanine, or canavanine-induced modifications in the machinery.


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 canavanine on intracellular localization of NP and cellular proteins. 143B cells infected for 2 h with VV-NP were incubated for an additional 6 h in the presence of 15 mM canavanine. Cells were fixed, permeabilized, and stained with the antibodies indicated. NP stained by anti-COOH antisera is shown in the right column. Gray-scale images on the left and right are merged in the middle column 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

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

Figure 7: Effect of canavanine on intracellular localization of NP and cellular proteins. 143B cells infected for 2 h with VV-NP were incubated for an additional 6 h in the presence of 15 mM canavanine. Cells were fixed, permeabilized, and stained with the antibodies indicated. NP stained by anti-COOH antisera is shown in the right column. Gray-scale images on the left and right are merged in the middle column with the color indicated by the text describing the antibody specificity. Arrows point to the MTOC. Bar, 10 μm.
Mentions: It was even possible to induce wild-type NP to localize to PODs and the MTOC by exposing VV-NP–infected to cells to canavanine, an amino acid analogue of Arg that induces protein misfolding (Fig. 7). NP in PODs and the MTOC was detected by anti-COOH but not anti-NH2 Abs, possibly due to the replacement of Arg in the NH2-terminal peptide with canavanine (the COOH peptide used for Ab generation does not contain Arg). In addition, large amounts of NP were now detected in the cytosol, an effect possibly related to canavanine modification of the nuclear localization signal. NP in PODs and MTOC recruited poly Ub, proteasomes, and HSC70. Notably this occurred in the absence of proteasome inhibitors, suggesting that the degradation machinery (which could also be affected by canavanine) was compromised under these conditions, either as a result of having to cope with vast quantities of proteins misfolded by incorporation of canavanine, or canavanine-induced modifications in the machinery.

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