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Chloroquine promotes apoptosis in melanoma cells by inhibiting BH3 domain-mediated PUMA degradation.

Lakhter AJ, Sahu RP, Sun Y, Kaufmann WK, Androphy EJ, Travers JB, Naidu SR - J. Invest. Dermatol. (2013)

Bottom Line: The Bcl homology-3 (BH3)-only protein p53 upregulated modulator of apoptosis (PUMA) counters Bcl-2 family anti-apoptotic proteins and promotes apoptosis.Fusion of the BH3 domain to a heterologous protein led to its rapid turnover that was inhibited by CQ.Although both CQ and inhibitors of lysosomal proteases stalled autophagy, only CQ stabilized PUMA protein and promoted apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN, USA.

ABSTRACT
The Bcl homology-3 (BH3)-only protein p53 upregulated modulator of apoptosis (PUMA) counters Bcl-2 family anti-apoptotic proteins and promotes apoptosis. Although PUMA is a key regulator of apoptosis, the post-transcriptional mechanisms that control PUMA protein stability are not understood. We show that a lysosome-independent activity of chloroquine (CQ) prevents degradation of PUMA protein, promotes apoptosis, and reduces the growth of melanoma xenografts in mice. Compared with wild-type PUMA, a BH3 domain-deleted PUMA protein showed impaired decay in melanoma cells. Fusion of the BH3 domain to a heterologous protein led to its rapid turnover that was inhibited by CQ. Although both CQ and inhibitors of lysosomal proteases stalled autophagy, only CQ stabilized PUMA protein and promoted apoptosis. Our results reveal a lysosomal protease-independent activity of CQ that selectively promotes apoptosis in melanoma cells.

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The BH3 domain of PUMA mediates its degradationa) To determine the role of the BH3 domain in PUMA protein stability, recombinant plasmids coding for HA-tagged wild-type or BH3 domain-deletion mutant PUMA were ectopically expressed in melanoma cells and the cell extracts were blotted with HA antibody or actin antibodies. b) Densitometry scanning of three experiments is shown on a graph. c) Schematic showing BH3 domain and its mutant peptides fusion with GFP. d) Plasmids from c were introduced into melanoma cells and the GFP expression were imaged. e) Mutant BH3 domain and wt-BH3 domain fused to GFP expressed in melanoma cells and the cells treated with CQ and blotted with indicated antibodies.
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Figure 5: The BH3 domain of PUMA mediates its degradationa) To determine the role of the BH3 domain in PUMA protein stability, recombinant plasmids coding for HA-tagged wild-type or BH3 domain-deletion mutant PUMA were ectopically expressed in melanoma cells and the cell extracts were blotted with HA antibody or actin antibodies. b) Densitometry scanning of three experiments is shown on a graph. c) Schematic showing BH3 domain and its mutant peptides fusion with GFP. d) Plasmids from c were introduced into melanoma cells and the GFP expression were imaged. e) Mutant BH3 domain and wt-BH3 domain fused to GFP expressed in melanoma cells and the cells treated with CQ and blotted with indicated antibodies.

Mentions: The BH3 domain of PUMA engages the hydrophobic groove of anti-apoptotic family members to activate apoptosis. Because this domain is critical for apoptosis, we reasoned that it may be involved in PUMA destabilization and that a mechanism in melanoma cells specifically may target this domain to avoid cell death. To test this idea, we introduced plasmids encoding for full-length or BH3-deletion mutant PUMA into melanoma cells and conducted cycloheximide chase assays for these ectopically expressed proteins. After 5 hours of cycloheximide incubation, nearly 50% of full-length PUMA protein remained (Fig 5a,b), while the BH3 domain deletion mutant protein levels were unchanged. These results suggest that the BH3 domain plays a critical role in PUMA protein destabilization. We questioned whether this BH3 domain was sufficient to direct degradation of a heterologous protein. We constructed GFP fusions with a BH3 domain or a mutant BH3 domain (mBH3) devoid of the conserved amino acid sequence Leu-Arg-Arg (Fig 5c). We introduced these recombinant plasmids into melanoma cells and examined GFP expression. No GFP signal was visualized with the wild-type BH3 fusion transfected cells (Fig 5d). In contrast, the mutant BH3 domain showed intense GFP fluorescence (Fig 5d). To further determine the effects of CQ on BH3 fusion protein stability, melanoma cells were transfected with these GFP constructs and treated with chloroquine. Immunoblots for GFP showed that chloroquine treatment restored the levels of wild-type BH3 domain fused GFP (Fig 5e). These results reinforce the essential role of the BH3 domain in PUMA degradation and that CQ inhibits degradation of PUMA actuated by this domain.


Chloroquine promotes apoptosis in melanoma cells by inhibiting BH3 domain-mediated PUMA degradation.

Lakhter AJ, Sahu RP, Sun Y, Kaufmann WK, Androphy EJ, Travers JB, Naidu SR - J. Invest. Dermatol. (2013)

The BH3 domain of PUMA mediates its degradationa) To determine the role of the BH3 domain in PUMA protein stability, recombinant plasmids coding for HA-tagged wild-type or BH3 domain-deletion mutant PUMA were ectopically expressed in melanoma cells and the cell extracts were blotted with HA antibody or actin antibodies. b) Densitometry scanning of three experiments is shown on a graph. c) Schematic showing BH3 domain and its mutant peptides fusion with GFP. d) Plasmids from c were introduced into melanoma cells and the GFP expression were imaged. e) Mutant BH3 domain and wt-BH3 domain fused to GFP expressed in melanoma cells and the cells treated with CQ and blotted with indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675185&req=5

Figure 5: The BH3 domain of PUMA mediates its degradationa) To determine the role of the BH3 domain in PUMA protein stability, recombinant plasmids coding for HA-tagged wild-type or BH3 domain-deletion mutant PUMA were ectopically expressed in melanoma cells and the cell extracts were blotted with HA antibody or actin antibodies. b) Densitometry scanning of three experiments is shown on a graph. c) Schematic showing BH3 domain and its mutant peptides fusion with GFP. d) Plasmids from c were introduced into melanoma cells and the GFP expression were imaged. e) Mutant BH3 domain and wt-BH3 domain fused to GFP expressed in melanoma cells and the cells treated with CQ and blotted with indicated antibodies.
Mentions: The BH3 domain of PUMA engages the hydrophobic groove of anti-apoptotic family members to activate apoptosis. Because this domain is critical for apoptosis, we reasoned that it may be involved in PUMA destabilization and that a mechanism in melanoma cells specifically may target this domain to avoid cell death. To test this idea, we introduced plasmids encoding for full-length or BH3-deletion mutant PUMA into melanoma cells and conducted cycloheximide chase assays for these ectopically expressed proteins. After 5 hours of cycloheximide incubation, nearly 50% of full-length PUMA protein remained (Fig 5a,b), while the BH3 domain deletion mutant protein levels were unchanged. These results suggest that the BH3 domain plays a critical role in PUMA protein destabilization. We questioned whether this BH3 domain was sufficient to direct degradation of a heterologous protein. We constructed GFP fusions with a BH3 domain or a mutant BH3 domain (mBH3) devoid of the conserved amino acid sequence Leu-Arg-Arg (Fig 5c). We introduced these recombinant plasmids into melanoma cells and examined GFP expression. No GFP signal was visualized with the wild-type BH3 fusion transfected cells (Fig 5d). In contrast, the mutant BH3 domain showed intense GFP fluorescence (Fig 5d). To further determine the effects of CQ on BH3 fusion protein stability, melanoma cells were transfected with these GFP constructs and treated with chloroquine. Immunoblots for GFP showed that chloroquine treatment restored the levels of wild-type BH3 domain fused GFP (Fig 5e). These results reinforce the essential role of the BH3 domain in PUMA degradation and that CQ inhibits degradation of PUMA actuated by this domain.

Bottom Line: The Bcl homology-3 (BH3)-only protein p53 upregulated modulator of apoptosis (PUMA) counters Bcl-2 family anti-apoptotic proteins and promotes apoptosis.Fusion of the BH3 domain to a heterologous protein led to its rapid turnover that was inhibited by CQ.Although both CQ and inhibitors of lysosomal proteases stalled autophagy, only CQ stabilized PUMA protein and promoted apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN, USA.

ABSTRACT
The Bcl homology-3 (BH3)-only protein p53 upregulated modulator of apoptosis (PUMA) counters Bcl-2 family anti-apoptotic proteins and promotes apoptosis. Although PUMA is a key regulator of apoptosis, the post-transcriptional mechanisms that control PUMA protein stability are not understood. We show that a lysosome-independent activity of chloroquine (CQ) prevents degradation of PUMA protein, promotes apoptosis, and reduces the growth of melanoma xenografts in mice. Compared with wild-type PUMA, a BH3 domain-deleted PUMA protein showed impaired decay in melanoma cells. Fusion of the BH3 domain to a heterologous protein led to its rapid turnover that was inhibited by CQ. Although both CQ and inhibitors of lysosomal proteases stalled autophagy, only CQ stabilized PUMA protein and promoted apoptosis. Our results reveal a lysosomal protease-independent activity of CQ that selectively promotes apoptosis in melanoma cells.

Show MeSH
Related in: MedlinePlus