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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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Related in: MedlinePlus

HAMLET inhibits proteasome activity.(A) Proteasome activity was monitored in vitro as chymotrypsin-dependent suc-LLVY-AMC cleavage (50 µM) over time and MG132 was used as proteasome inhibitor. Except for the first 10 minutes HAMLET reduced proteasome activity compared to the control. (B) Inhibition of proteasome activity in A549 cells. Cytoplasmic extracts were prepared from cells treated with HAMLET or native α-lactalbumin (34 µM) after 1, 3 and 6 hours of incubation and enzymatic activity was quantified by suc-LLVY-AMC cleavage (50 µM). MG132 was used to inhibit proteasome function.
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pone-0005229-g004: HAMLET inhibits proteasome activity.(A) Proteasome activity was monitored in vitro as chymotrypsin-dependent suc-LLVY-AMC cleavage (50 µM) over time and MG132 was used as proteasome inhibitor. Except for the first 10 minutes HAMLET reduced proteasome activity compared to the control. (B) Inhibition of proteasome activity in A549 cells. Cytoplasmic extracts were prepared from cells treated with HAMLET or native α-lactalbumin (34 µM) after 1, 3 and 6 hours of incubation and enzymatic activity was quantified by suc-LLVY-AMC cleavage (50 µM). MG132 was used to inhibit proteasome function.

Mentions: To examine if HAMLET influenced proteasome function, we quantified the proteasome activity in vitro, using isolated 20S proteasomes (200 ng) and chromogenic substrates (LLVY-AMC, 50 µM). The proteasomes were enzymatically active as shown by substrate cleavage in the control and this activity was inhibited by MG132 (50 µM, Figure 4A). HAMLET caused a low, initial burst of proteasome activity lasting about 10 minutes but subsequently HAMLET acted as a partial proteasome inhibitor compared to the control (Figure 4A). The results indicated that HAMLET impaired the catalytic activity of the proteasome core. This effect was confirmed in extracts from HAMLET-treated A549 carcinoma cells (34 µM, 1, 3 and 6 hours). A significant reduction in proteasome activity was detected after 3 and 6 hours of HAMLET treatment (Figure 4B, p<0.01, 6 hours).


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)

HAMLET inhibits proteasome activity.(A) Proteasome activity was monitored in vitro as chymotrypsin-dependent suc-LLVY-AMC cleavage (50 µM) over time and MG132 was used as proteasome inhibitor. Except for the first 10 minutes HAMLET reduced proteasome activity compared to the control. (B) Inhibition of proteasome activity in A549 cells. Cytoplasmic extracts were prepared from cells treated with HAMLET or native α-lactalbumin (34 µM) after 1, 3 and 6 hours of incubation and enzymatic activity was quantified by suc-LLVY-AMC cleavage (50 µM). MG132 was used to inhibit proteasome function.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664966&req=5

pone-0005229-g004: HAMLET inhibits proteasome activity.(A) Proteasome activity was monitored in vitro as chymotrypsin-dependent suc-LLVY-AMC cleavage (50 µM) over time and MG132 was used as proteasome inhibitor. Except for the first 10 minutes HAMLET reduced proteasome activity compared to the control. (B) Inhibition of proteasome activity in A549 cells. Cytoplasmic extracts were prepared from cells treated with HAMLET or native α-lactalbumin (34 µM) after 1, 3 and 6 hours of incubation and enzymatic activity was quantified by suc-LLVY-AMC cleavage (50 µM). MG132 was used to inhibit proteasome function.
Mentions: To examine if HAMLET influenced proteasome function, we quantified the proteasome activity in vitro, using isolated 20S proteasomes (200 ng) and chromogenic substrates (LLVY-AMC, 50 µM). The proteasomes were enzymatically active as shown by substrate cleavage in the control and this activity was inhibited by MG132 (50 µM, Figure 4A). HAMLET caused a low, initial burst of proteasome activity lasting about 10 minutes but subsequently HAMLET acted as a partial proteasome inhibitor compared to the control (Figure 4A). The results indicated that HAMLET impaired the catalytic activity of the proteasome core. This effect was confirmed in extracts from HAMLET-treated A549 carcinoma cells (34 µM, 1, 3 and 6 hours). A significant reduction in proteasome activity was detected after 3 and 6 hours of HAMLET treatment (Figure 4B, p<0.01, 6 hours).

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