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Changes in proteasome structure and function caused by HAMLET in tumor cells.

Gustafsson L, Aits S, Onnerfjord P, Trulsson M, Storm P, Svanborg C - PLoS ONE (2009)

Bottom Line: HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin.The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure.We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

ABSTRACT

Background: Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death.

Methodology/principal findings: HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells.

Conclusions/significance: The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

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Changes in 20S proteasome staining after HAMLET treatment of carcinoma cells.(A–C) Changes in the staining of 20S proteasome subunits in A549 cells after HAMLET treatment (34 and 45 µM, 1 hour). Cells were stained with antibodies against the 20S proteasome core subunits (α5, α7, β1, β5, β5i and β7), antibodies against the β1 subunit or antibodies against the β5 subunit. (A) Confocal images showing increased core and β1 subunit staining, and decreased β5 subunit staining after HAMLET treatment (45 µM, 1 hour). (B) Fluorescence intensity was quantified in control and HAMLET-treated cells (34 and 45 µM, 1 hour). (C) The increase in core subunit staining after HAMLET was blocked by proteasome inhibition with MG132. (D) Western blot of cell extracts from cells treated with HAMLET (34 µM) for 1 and 6 hours, showing loss of 20S proteasomes staining after 6 hours. Antibodies against the 20S proteasome core subunits were used.
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pone-0005229-g006: Changes in 20S proteasome staining after HAMLET treatment of carcinoma cells.(A–C) Changes in the staining of 20S proteasome subunits in A549 cells after HAMLET treatment (34 and 45 µM, 1 hour). Cells were stained with antibodies against the 20S proteasome core subunits (α5, α7, β1, β5, β5i and β7), antibodies against the β1 subunit or antibodies against the β5 subunit. (A) Confocal images showing increased core and β1 subunit staining, and decreased β5 subunit staining after HAMLET treatment (45 µM, 1 hour). (B) Fluorescence intensity was quantified in control and HAMLET-treated cells (34 and 45 µM, 1 hour). (C) The increase in core subunit staining after HAMLET was blocked by proteasome inhibition with MG132. (D) Western blot of cell extracts from cells treated with HAMLET (34 µM) for 1 and 6 hours, showing loss of 20S proteasomes staining after 6 hours. Antibodies against the 20S proteasome core subunits were used.

Mentions: Confocal microscopy was used to visualize the change in 20S proteasomes structure, following HAMLET treatment. A increase in staining of 20S proteasome core subunits was observed after HAMLET treatment suggesting that the epitopes targeted by the polyclonal antibodies became more accessible (Figure 6A, B). This effect was inhibited by MG132 (Figure 6C). The effect was specific for HAMLET, as α-lactalbumin treatment did not alter proteasome staining (data not shown). A similar increase in staining after HAMLET treatment was observed using anti-β1 subunit antibodies (Figure 6A, B). In contrast, when using the polyclonal β5-subunit antibody, we observed a decrease in staining consistent with a loss of this subunit after HAMLET treatment (Figure 6A, B).


Changes in proteasome structure and function caused by HAMLET in tumor cells.

Gustafsson L, Aits S, Onnerfjord P, Trulsson M, Storm P, Svanborg C - PLoS ONE (2009)

Changes in 20S proteasome staining after HAMLET treatment of carcinoma cells.(A–C) Changes in the staining of 20S proteasome subunits in A549 cells after HAMLET treatment (34 and 45 µM, 1 hour). Cells were stained with antibodies against the 20S proteasome core subunits (α5, α7, β1, β5, β5i and β7), antibodies against the β1 subunit or antibodies against the β5 subunit. (A) Confocal images showing increased core and β1 subunit staining, and decreased β5 subunit staining after HAMLET treatment (45 µM, 1 hour). (B) Fluorescence intensity was quantified in control and HAMLET-treated cells (34 and 45 µM, 1 hour). (C) The increase in core subunit staining after HAMLET was blocked by proteasome inhibition with MG132. (D) Western blot of cell extracts from cells treated with HAMLET (34 µM) for 1 and 6 hours, showing loss of 20S proteasomes staining after 6 hours. Antibodies against the 20S proteasome core subunits were used.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664966&req=5

pone-0005229-g006: Changes in 20S proteasome staining after HAMLET treatment of carcinoma cells.(A–C) Changes in the staining of 20S proteasome subunits in A549 cells after HAMLET treatment (34 and 45 µM, 1 hour). Cells were stained with antibodies against the 20S proteasome core subunits (α5, α7, β1, β5, β5i and β7), antibodies against the β1 subunit or antibodies against the β5 subunit. (A) Confocal images showing increased core and β1 subunit staining, and decreased β5 subunit staining after HAMLET treatment (45 µM, 1 hour). (B) Fluorescence intensity was quantified in control and HAMLET-treated cells (34 and 45 µM, 1 hour). (C) The increase in core subunit staining after HAMLET was blocked by proteasome inhibition with MG132. (D) Western blot of cell extracts from cells treated with HAMLET (34 µM) for 1 and 6 hours, showing loss of 20S proteasomes staining after 6 hours. Antibodies against the 20S proteasome core subunits were used.
Mentions: Confocal microscopy was used to visualize the change in 20S proteasomes structure, following HAMLET treatment. A increase in staining of 20S proteasome core subunits was observed after HAMLET treatment suggesting that the epitopes targeted by the polyclonal antibodies became more accessible (Figure 6A, B). This effect was inhibited by MG132 (Figure 6C). The effect was specific for HAMLET, as α-lactalbumin treatment did not alter proteasome staining (data not shown). A similar increase in staining after HAMLET treatment was observed using anti-β1 subunit antibodies (Figure 6A, B). In contrast, when using the polyclonal β5-subunit antibody, we observed a decrease in staining consistent with a loss of this subunit after HAMLET treatment (Figure 6A, B).

Bottom Line: HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin.The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure.We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

ABSTRACT

Background: Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death.

Methodology/principal findings: HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells.

Conclusions/significance: The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

Show MeSH
Related in: MedlinePlus