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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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HAMLET is a complex of partially unfolded α-lactalbumin and oleic acid.(A) HAMLET is formed when native α-lactalbumin releases the strongly bound Ca2+ and exposes a fatty-acid binding site, which permits oleic acid (C18:1, 9 cis) to interact and stabilize the partially unfolded state. The figure was generated using the crystal structure of human α-lactalbumin [39] and MOLMOL 2K.2 [40]. (B) Near-UV CD spectra were recorded in sodium phosphate buffer without EDTA. Native α-lactalbumin showed the expected minimum at 270 nm arising from the tyrosine and a maximum at 293 nm arising from the tryptophan residues. In HAMLET, α-lactalbumin showed a loss of signal at 270 nm and at 293 nm, typical of partial unfolding [9], [16]. Apo α-lactalbumin was obtained by EDTA treatment and showed a similar loss of signal. (C) Effect of HAMLET on tumor cells and healthy cells. Glioblastoma cells and kidney carcinoma cells treated with HAMLET (34 µM, 1 and 3 hours) died while normal astrocytes and renal epithelial cells (HRTEC) remained viable. (D) Difference in uptake of HAMLET and α-lactalbumin by tumor cells. Lung carcinoma cells (A549) were exposed to 34 µM of Alexa Fluor 568-labeled HAMLET or α-lactalbumin (both red). (E) Quantification by flow cytometry of HAMLET or α-lactalbumin uptake by A549 cells (N = 10,000) after 1 and 3 hours of incubation.
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pone-0005229-g001: HAMLET is a complex of partially unfolded α-lactalbumin and oleic acid.(A) HAMLET is formed when native α-lactalbumin releases the strongly bound Ca2+ and exposes a fatty-acid binding site, which permits oleic acid (C18:1, 9 cis) to interact and stabilize the partially unfolded state. The figure was generated using the crystal structure of human α-lactalbumin [39] and MOLMOL 2K.2 [40]. (B) Near-UV CD spectra were recorded in sodium phosphate buffer without EDTA. Native α-lactalbumin showed the expected minimum at 270 nm arising from the tyrosine and a maximum at 293 nm arising from the tryptophan residues. In HAMLET, α-lactalbumin showed a loss of signal at 270 nm and at 293 nm, typical of partial unfolding [9], [16]. Apo α-lactalbumin was obtained by EDTA treatment and showed a similar loss of signal. (C) Effect of HAMLET on tumor cells and healthy cells. Glioblastoma cells and kidney carcinoma cells treated with HAMLET (34 µM, 1 and 3 hours) died while normal astrocytes and renal epithelial cells (HRTEC) remained viable. (D) Difference in uptake of HAMLET and α-lactalbumin by tumor cells. Lung carcinoma cells (A549) were exposed to 34 µM of Alexa Fluor 568-labeled HAMLET or α-lactalbumin (both red). (E) Quantification by flow cytometry of HAMLET or α-lactalbumin uptake by A549 cells (N = 10,000) after 1 and 3 hours of incubation.

Mentions: α-Lactalbumin is secreted by the mammary gland as a mature, folded protein but can unfold by releasing Ca2+ [22]–[24]. Extracellular unfolding can occur for example at low pH [25] in environments like the stomach. To produce the HAMLET complex in the laboratory, native α-lactalbumin is partially unfolded with EDTA to the apo state and then converted to HAMLET on an oleic acid-preconditioned ion exchange matrix (Figure 1A) [9]. The fatty acid stabilizes the protein in the partially unfolded state, as shown by near-UV CD spectroscopy (Figure 1B) [9].


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 is a complex of partially unfolded α-lactalbumin and oleic acid.(A) HAMLET is formed when native α-lactalbumin releases the strongly bound Ca2+ and exposes a fatty-acid binding site, which permits oleic acid (C18:1, 9 cis) to interact and stabilize the partially unfolded state. The figure was generated using the crystal structure of human α-lactalbumin [39] and MOLMOL 2K.2 [40]. (B) Near-UV CD spectra were recorded in sodium phosphate buffer without EDTA. Native α-lactalbumin showed the expected minimum at 270 nm arising from the tyrosine and a maximum at 293 nm arising from the tryptophan residues. In HAMLET, α-lactalbumin showed a loss of signal at 270 nm and at 293 nm, typical of partial unfolding [9], [16]. Apo α-lactalbumin was obtained by EDTA treatment and showed a similar loss of signal. (C) Effect of HAMLET on tumor cells and healthy cells. Glioblastoma cells and kidney carcinoma cells treated with HAMLET (34 µM, 1 and 3 hours) died while normal astrocytes and renal epithelial cells (HRTEC) remained viable. (D) Difference in uptake of HAMLET and α-lactalbumin by tumor cells. Lung carcinoma cells (A549) were exposed to 34 µM of Alexa Fluor 568-labeled HAMLET or α-lactalbumin (both red). (E) Quantification by flow cytometry of HAMLET or α-lactalbumin uptake by A549 cells (N = 10,000) after 1 and 3 hours of incubation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005229-g001: HAMLET is a complex of partially unfolded α-lactalbumin and oleic acid.(A) HAMLET is formed when native α-lactalbumin releases the strongly bound Ca2+ and exposes a fatty-acid binding site, which permits oleic acid (C18:1, 9 cis) to interact and stabilize the partially unfolded state. The figure was generated using the crystal structure of human α-lactalbumin [39] and MOLMOL 2K.2 [40]. (B) Near-UV CD spectra were recorded in sodium phosphate buffer without EDTA. Native α-lactalbumin showed the expected minimum at 270 nm arising from the tyrosine and a maximum at 293 nm arising from the tryptophan residues. In HAMLET, α-lactalbumin showed a loss of signal at 270 nm and at 293 nm, typical of partial unfolding [9], [16]. Apo α-lactalbumin was obtained by EDTA treatment and showed a similar loss of signal. (C) Effect of HAMLET on tumor cells and healthy cells. Glioblastoma cells and kidney carcinoma cells treated with HAMLET (34 µM, 1 and 3 hours) died while normal astrocytes and renal epithelial cells (HRTEC) remained viable. (D) Difference in uptake of HAMLET and α-lactalbumin by tumor cells. Lung carcinoma cells (A549) were exposed to 34 µM of Alexa Fluor 568-labeled HAMLET or α-lactalbumin (both red). (E) Quantification by flow cytometry of HAMLET or α-lactalbumin uptake by A549 cells (N = 10,000) after 1 and 3 hours of incubation.
Mentions: α-Lactalbumin is secreted by the mammary gland as a mature, folded protein but can unfold by releasing Ca2+ [22]–[24]. Extracellular unfolding can occur for example at low pH [25] in environments like the stomach. To produce the HAMLET complex in the laboratory, native α-lactalbumin is partially unfolded with EDTA to the apo state and then converted to HAMLET on an oleic acid-preconditioned ion exchange matrix (Figure 1A) [9]. The fatty acid stabilizes the protein in the partially unfolded state, as shown by near-UV CD spectroscopy (Figure 1B) [9].

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