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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

Proteasome-dependent degradation of dNPpep. 143B cells were pulse radiolabeled with [35S]Met and chased for up to 120 min at 37°C in the presence or absence of LC. Radioactive proteins soluble in 1% TX100 (sol) or insoluble (ins) were separated by SDS-PAGE and the bands corresponding to dNPpep or NPpep located in the dried gel (a) and quantitated (b) after normalization using a VV-encoded protein as an internal standard (indicated as VV).
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Figure 2: Proteasome-dependent degradation of dNPpep. 143B cells were pulse radiolabeled with [35S]Met and chased for up to 120 min at 37°C in the presence or absence of LC. Radioactive proteins soluble in 1% TX100 (sol) or insoluble (ins) were separated by SDS-PAGE and the bands corresponding to dNPpep or NPpep located in the dried gel (a) and quantitated (b) after normalization using a VV-encoded protein as an internal standard (indicated as VV).

Mentions: 143 cells infected 3 h previously were incubated in Met-free DMEM with 100 μM lactacystin (LC) for 40 min, and then radiolabeled by 5 min of incubation with [35S]Met. After washing, cells were chased at 37°C for up to 120 min in Met containing DMEM. At appropriate times, 2 × 106 cells were removed to ice. Cells were incubated with 1% (vol/vol) Triton X-100 (TX100) containing buffer and centrifuged at 15,000 g for 10 min. Supernatants and pellets were suspended in boiling SDS-PAGE sample buffer boiled for 5 min, and analyzed by SDS-PAGE. Images of autoradiographs of the dried gels were digitized by a flat bed scanner, assembled using Adobe Photoshop software and printed with a Fujix Pictrography digital printer. The radioactivity present in dried gels was quantitated using a PhosphorImager (Molecular Devices) and the screens supplied by the manufacturer. The VV protein shown in Fig. 2 a was used as an internal standard for normalization of the amount of protein recovered from each sample.


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 degradation of dNPpep. 143B cells were pulse radiolabeled with [35S]Met and chased for up to 120 min at 37°C in the presence or absence of LC. Radioactive proteins soluble in 1% TX100 (sol) or insoluble (ins) were separated by SDS-PAGE and the bands corresponding to dNPpep or NPpep located in the dried gel (a) and quantitated (b) after normalization using a VV-encoded protein as an internal standard (indicated as VV).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Proteasome-dependent degradation of dNPpep. 143B cells were pulse radiolabeled with [35S]Met and chased for up to 120 min at 37°C in the presence or absence of LC. Radioactive proteins soluble in 1% TX100 (sol) or insoluble (ins) were separated by SDS-PAGE and the bands corresponding to dNPpep or NPpep located in the dried gel (a) and quantitated (b) after normalization using a VV-encoded protein as an internal standard (indicated as VV).
Mentions: 143 cells infected 3 h previously were incubated in Met-free DMEM with 100 μM lactacystin (LC) for 40 min, and then radiolabeled by 5 min of incubation with [35S]Met. After washing, cells were chased at 37°C for up to 120 min in Met containing DMEM. At appropriate times, 2 × 106 cells were removed to ice. Cells were incubated with 1% (vol/vol) Triton X-100 (TX100) containing buffer and centrifuged at 15,000 g for 10 min. Supernatants and pellets were suspended in boiling SDS-PAGE sample buffer boiled for 5 min, and analyzed by SDS-PAGE. Images of autoradiographs of the dried gels were digitized by a flat bed scanner, assembled using Adobe Photoshop software and printed with a Fujix Pictrography digital printer. The radioactivity present in dried gels was quantitated using a PhosphorImager (Molecular Devices) and the screens supplied by the manufacturer. The VV protein shown in Fig. 2 a was used as an internal standard for normalization of the amount of protein recovered from each sample.

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