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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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Related in: MedlinePlus

Fractionation and biochemical characterization of dNPpep. (A) Extracts from rVV-infected 143B cells incubated with or without 20 μM zLLL were analyzed by Western blotting using anti-NH2 antibodies. Note that the panel on the left is from a different gel than that on the right, and that the corresponding bands are more compressed. (B) HeLa cells infected with the rVV indicated for 90 min were incubated for an additional 3.5 h in the presence of 40 μM zLLL. Total cell extracts were analyzed by Western blotting using antibodies against the NH2- or COOH-terminal peptides. Calculated Mr of the lower mobility bands indicated in red. (C) 143B cells expressing dNPpep (left) or NPpep (right) were extracted sequentially by NP-40, DNase I digestion, 2 M NaCl, DNase I/RNAase digestion, and then paraformaldehyde fixed, stained with anti-PML (top) or anti-NH2 antibodies, and analyzed using the LCSM. Gray-scale images on the top and bottom are merged in the middle with the color indicated by the text describing the antibody specificity. The arrow points to the MTOC. (D) 143B cells infected with a control virus (C), NPpep (N), or dNPpep (D) were labeled for 5 min with [35S]Met and chased for 40 min. Cells were extracted as above, and acetone precipitates were analyzed by SDS-PAGE and the radiolabeled proteins visualized using a PhosphorImager. Shown are the regions of the gel containing the proteins of interest along with control cellular and VV proteins. The intensity of total and NP-40 lysates was reduced fourfold before the 16–8 bit digital conversion to enable visualization of individual protein bands in all of the fractions. Arrows indicate NPpep and dNPpep. Based on PhosphorImager quantitation (and taking into account sample recovery), 2.3-fold more NPpep is present in total lysates than dNPpep, whereas 6.8-fold more dNPpep is recovered in the Empigen BB step.
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Figure 4: Fractionation and biochemical characterization of dNPpep. (A) Extracts from rVV-infected 143B cells incubated with or without 20 μM zLLL were analyzed by Western blotting using anti-NH2 antibodies. Note that the panel on the left is from a different gel than that on the right, and that the corresponding bands are more compressed. (B) HeLa cells infected with the rVV indicated for 90 min were incubated for an additional 3.5 h in the presence of 40 μM zLLL. Total cell extracts were analyzed by Western blotting using antibodies against the NH2- or COOH-terminal peptides. Calculated Mr of the lower mobility bands indicated in red. (C) 143B cells expressing dNPpep (left) or NPpep (right) were extracted sequentially by NP-40, DNase I digestion, 2 M NaCl, DNase I/RNAase digestion, and then paraformaldehyde fixed, stained with anti-PML (top) or anti-NH2 antibodies, and analyzed using the LCSM. Gray-scale images on the top and bottom are merged in the middle with the color indicated by the text describing the antibody specificity. The arrow points to the MTOC. (D) 143B cells infected with a control virus (C), NPpep (N), or dNPpep (D) were labeled for 5 min with [35S]Met and chased for 40 min. Cells were extracted as above, and acetone precipitates were analyzed by SDS-PAGE and the radiolabeled proteins visualized using a PhosphorImager. Shown are the regions of the gel containing the proteins of interest along with control cellular and VV proteins. The intensity of total and NP-40 lysates was reduced fourfold before the 16–8 bit digital conversion to enable visualization of individual protein bands in all of the fractions. Arrows indicate NPpep and dNPpep. Based on PhosphorImager quantitation (and taking into account sample recovery), 2.3-fold more NPpep is present in total lysates than dNPpep, whereas 6.8-fold more dNPpep is recovered in the Empigen BB step.

Mentions: PODs can be partially purified by progressive extraction designed to isolate the nuclear matrix (Staufenbiel and Deppert 1984). This was performed biochemically and cytoimmunochemically. rVV-infected 143B cells incubated with zLLL were subjected sequentially to NP-40, DNase I, high salt, DNase I/RNAase, and then fixed with paraformaldehyde and examined using the LCSM after staining with anti-NH2 and anti-POD antibodies. Under these conditions dNPpep was easily detected in PODs and the MTOC (Fig. 4 C, arrow), whereas the low level staining of remaining NPpep was in a pattern not clearly related to PODs.


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)

Fractionation and biochemical characterization of dNPpep. (A) Extracts from rVV-infected 143B cells incubated with or without 20 μM zLLL were analyzed by Western blotting using anti-NH2 antibodies. Note that the panel on the left is from a different gel than that on the right, and that the corresponding bands are more compressed. (B) HeLa cells infected with the rVV indicated for 90 min were incubated for an additional 3.5 h in the presence of 40 μM zLLL. Total cell extracts were analyzed by Western blotting using antibodies against the NH2- or COOH-terminal peptides. Calculated Mr of the lower mobility bands indicated in red. (C) 143B cells expressing dNPpep (left) or NPpep (right) were extracted sequentially by NP-40, DNase I digestion, 2 M NaCl, DNase I/RNAase digestion, and then paraformaldehyde fixed, stained with anti-PML (top) or anti-NH2 antibodies, and analyzed using the LCSM. Gray-scale images on the top and bottom are merged in the middle with the color indicated by the text describing the antibody specificity. The arrow points to the MTOC. (D) 143B cells infected with a control virus (C), NPpep (N), or dNPpep (D) were labeled for 5 min with [35S]Met and chased for 40 min. Cells were extracted as above, and acetone precipitates were analyzed by SDS-PAGE and the radiolabeled proteins visualized using a PhosphorImager. Shown are the regions of the gel containing the proteins of interest along with control cellular and VV proteins. The intensity of total and NP-40 lysates was reduced fourfold before the 16–8 bit digital conversion to enable visualization of individual protein bands in all of the fractions. Arrows indicate NPpep and dNPpep. Based on PhosphorImager quantitation (and taking into account sample recovery), 2.3-fold more NPpep is present in total lysates than dNPpep, whereas 6.8-fold more dNPpep is recovered in the Empigen BB step.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199725&req=5

Figure 4: Fractionation and biochemical characterization of dNPpep. (A) Extracts from rVV-infected 143B cells incubated with or without 20 μM zLLL were analyzed by Western blotting using anti-NH2 antibodies. Note that the panel on the left is from a different gel than that on the right, and that the corresponding bands are more compressed. (B) HeLa cells infected with the rVV indicated for 90 min were incubated for an additional 3.5 h in the presence of 40 μM zLLL. Total cell extracts were analyzed by Western blotting using antibodies against the NH2- or COOH-terminal peptides. Calculated Mr of the lower mobility bands indicated in red. (C) 143B cells expressing dNPpep (left) or NPpep (right) were extracted sequentially by NP-40, DNase I digestion, 2 M NaCl, DNase I/RNAase digestion, and then paraformaldehyde fixed, stained with anti-PML (top) or anti-NH2 antibodies, and analyzed using the LCSM. Gray-scale images on the top and bottom are merged in the middle with the color indicated by the text describing the antibody specificity. The arrow points to the MTOC. (D) 143B cells infected with a control virus (C), NPpep (N), or dNPpep (D) were labeled for 5 min with [35S]Met and chased for 40 min. Cells were extracted as above, and acetone precipitates were analyzed by SDS-PAGE and the radiolabeled proteins visualized using a PhosphorImager. Shown are the regions of the gel containing the proteins of interest along with control cellular and VV proteins. The intensity of total and NP-40 lysates was reduced fourfold before the 16–8 bit digital conversion to enable visualization of individual protein bands in all of the fractions. Arrows indicate NPpep and dNPpep. Based on PhosphorImager quantitation (and taking into account sample recovery), 2.3-fold more NPpep is present in total lysates than dNPpep, whereas 6.8-fold more dNPpep is recovered in the Empigen BB step.
Mentions: PODs can be partially purified by progressive extraction designed to isolate the nuclear matrix (Staufenbiel and Deppert 1984). This was performed biochemically and cytoimmunochemically. rVV-infected 143B cells incubated with zLLL were subjected sequentially to NP-40, DNase I, high salt, DNase I/RNAase, and then fixed with paraformaldehyde and examined using the LCSM after staining with anti-NH2 and anti-POD antibodies. Under these conditions dNPpep was easily detected in PODs and the MTOC (Fig. 4 C, arrow), whereas the low level staining of remaining NPpep was in a pattern not clearly related to PODs.

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