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The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein.

Shi C, Huang X, Zhang B, Zhu D, Luo H, Lu Q, Xiong WC, Mei L, Luo S - PLoS ONE (2015)

Bottom Line: Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs).Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo.The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP).

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China.

ABSTRACT

Background: Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear.

Results: In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells.

Conclusions: Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD.

No MeSH data available.


Related in: MedlinePlus

The inhibition of HSP90 reduces the aggregation and cell death caused by mutant A17-PABPN1.(A, B) 17-AAG decreases PABPN1-induced INIs in myotubes. C2C12 myoblasts were transfected with 0.5 mg GFP-PABPN1 constructs. Cells were switched to differentiation medium 24 hr after transfection, and fully differentiated myotubes were incubated with 1 μM 17-AAG for 12 hr; EGFP-positive myotubes were scored. In some experiments, the myotubes were treated with 1 M KCl at room temperature for 5 min to serve as a positive control. **, P < 0.01, n = 5. (C, D) The formation of A17-PABPN1 aggregates is impaired in 17-AAG treated HeLa cells. HeLa cells were transfected with GFP-tagged A17-PABPN1 constructs. Thirty-six hours post-transfection, the cells were incubated with or without 1 μM 17-AAG for 48 hr. The arrows identify cells containing INIs (C). Aggregate formation was analyzed with ZEN 2010 software (~500 transfected cells in each case). The results are the means ± SEM of three independent experiments (D). **, P < 0.01. (E) 17-AAG protects against mutant PABPN1 toxicity in C2C12 cells. C2C12 myoblasts were transfected with A10-PABPN1 and A17-PABPN1. Cells were harvested and replated in 96-well tissue culture plates, and the cells were treated with 500 nM 17-AAG for two days. Cell viability was measured by MTT assay. Data are mean ± SEM of three independent experiments. N.S., no significance; **, P < 0.01.
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pone.0138936.g004: The inhibition of HSP90 reduces the aggregation and cell death caused by mutant A17-PABPN1.(A, B) 17-AAG decreases PABPN1-induced INIs in myotubes. C2C12 myoblasts were transfected with 0.5 mg GFP-PABPN1 constructs. Cells were switched to differentiation medium 24 hr after transfection, and fully differentiated myotubes were incubated with 1 μM 17-AAG for 12 hr; EGFP-positive myotubes were scored. In some experiments, the myotubes were treated with 1 M KCl at room temperature for 5 min to serve as a positive control. **, P < 0.01, n = 5. (C, D) The formation of A17-PABPN1 aggregates is impaired in 17-AAG treated HeLa cells. HeLa cells were transfected with GFP-tagged A17-PABPN1 constructs. Thirty-six hours post-transfection, the cells were incubated with or without 1 μM 17-AAG for 48 hr. The arrows identify cells containing INIs (C). Aggregate formation was analyzed with ZEN 2010 software (~500 transfected cells in each case). The results are the means ± SEM of three independent experiments (D). **, P < 0.01. (E) 17-AAG protects against mutant PABPN1 toxicity in C2C12 cells. C2C12 myoblasts were transfected with A10-PABPN1 and A17-PABPN1. Cells were harvested and replated in 96-well tissue culture plates, and the cells were treated with 500 nM 17-AAG for two days. Cell viability was measured by MTT assay. Data are mean ± SEM of three independent experiments. N.S., no significance; **, P < 0.01.

Mentions: To explore the biological significance of the effect of 17-AAG on A17-PABPN1, we studied aggregate formation in A17-PABPN1-transfected C2C12 myotubes, a cellular model of OPMD [33]. Myotubes were transfected with A17-PABPN1 or A10-PABPN1, treated with 17-AAG and examined for INIs. PABPN1-positive INIs were observed in myotubes expressing either A10-PABPN1 or A17-PABPN1 as shown in (Fig 4A). The INIs that formed in A10-PABPN1-expressing cells were soluble in 1 M KCl whereas A17-PABPN1-positive INIs were resistant to KCl treatment (Fig 4B), which is in agreement with previous observations [32, 33]. Interestingly, the number of INIs formed by A17-PABPN1 was reduced in myotubes treated with 17-AAG, suggesting that mutant A17-PABPN1 is degraded by the 17-AAG treatment. To obtain additional evidence to support this hypothesis, we examined aggregate formation in A17-PABPN1-transfected HeLa cells in response to 17-AAG treatment alone. As shown in Fig 4C and 4D, A17-PABPN1-transfected HeLa cells that form large, visible aggregates in response to DMSO showed a reduction in the size and intensity of these aggregates in response to 17-AAG treatment (2 μM for 24 hr). To determine whether treatment with 17-AAG protects against PABPN1 toxicity, we transfected C2C12 cells with wild-type A10-PABPN1 and mutant A17-PABPN1 and studied the effects on cell viability. We found that 17-AAG (100 nM, 48 hr) significantly protected C2C12 cells against mutant A17-PABPN1-induced cell death (Fig 4E).


The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein.

Shi C, Huang X, Zhang B, Zhu D, Luo H, Lu Q, Xiong WC, Mei L, Luo S - PLoS ONE (2015)

The inhibition of HSP90 reduces the aggregation and cell death caused by mutant A17-PABPN1.(A, B) 17-AAG decreases PABPN1-induced INIs in myotubes. C2C12 myoblasts were transfected with 0.5 mg GFP-PABPN1 constructs. Cells were switched to differentiation medium 24 hr after transfection, and fully differentiated myotubes were incubated with 1 μM 17-AAG for 12 hr; EGFP-positive myotubes were scored. In some experiments, the myotubes were treated with 1 M KCl at room temperature for 5 min to serve as a positive control. **, P < 0.01, n = 5. (C, D) The formation of A17-PABPN1 aggregates is impaired in 17-AAG treated HeLa cells. HeLa cells were transfected with GFP-tagged A17-PABPN1 constructs. Thirty-six hours post-transfection, the cells were incubated with or without 1 μM 17-AAG for 48 hr. The arrows identify cells containing INIs (C). Aggregate formation was analyzed with ZEN 2010 software (~500 transfected cells in each case). The results are the means ± SEM of three independent experiments (D). **, P < 0.01. (E) 17-AAG protects against mutant PABPN1 toxicity in C2C12 cells. C2C12 myoblasts were transfected with A10-PABPN1 and A17-PABPN1. Cells were harvested and replated in 96-well tissue culture plates, and the cells were treated with 500 nM 17-AAG for two days. Cell viability was measured by MTT assay. Data are mean ± SEM of three independent experiments. N.S., no significance; **, P < 0.01.
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pone.0138936.g004: The inhibition of HSP90 reduces the aggregation and cell death caused by mutant A17-PABPN1.(A, B) 17-AAG decreases PABPN1-induced INIs in myotubes. C2C12 myoblasts were transfected with 0.5 mg GFP-PABPN1 constructs. Cells were switched to differentiation medium 24 hr after transfection, and fully differentiated myotubes were incubated with 1 μM 17-AAG for 12 hr; EGFP-positive myotubes were scored. In some experiments, the myotubes were treated with 1 M KCl at room temperature for 5 min to serve as a positive control. **, P < 0.01, n = 5. (C, D) The formation of A17-PABPN1 aggregates is impaired in 17-AAG treated HeLa cells. HeLa cells were transfected with GFP-tagged A17-PABPN1 constructs. Thirty-six hours post-transfection, the cells were incubated with or without 1 μM 17-AAG for 48 hr. The arrows identify cells containing INIs (C). Aggregate formation was analyzed with ZEN 2010 software (~500 transfected cells in each case). The results are the means ± SEM of three independent experiments (D). **, P < 0.01. (E) 17-AAG protects against mutant PABPN1 toxicity in C2C12 cells. C2C12 myoblasts were transfected with A10-PABPN1 and A17-PABPN1. Cells were harvested and replated in 96-well tissue culture plates, and the cells were treated with 500 nM 17-AAG for two days. Cell viability was measured by MTT assay. Data are mean ± SEM of three independent experiments. N.S., no significance; **, P < 0.01.
Mentions: To explore the biological significance of the effect of 17-AAG on A17-PABPN1, we studied aggregate formation in A17-PABPN1-transfected C2C12 myotubes, a cellular model of OPMD [33]. Myotubes were transfected with A17-PABPN1 or A10-PABPN1, treated with 17-AAG and examined for INIs. PABPN1-positive INIs were observed in myotubes expressing either A10-PABPN1 or A17-PABPN1 as shown in (Fig 4A). The INIs that formed in A10-PABPN1-expressing cells were soluble in 1 M KCl whereas A17-PABPN1-positive INIs were resistant to KCl treatment (Fig 4B), which is in agreement with previous observations [32, 33]. Interestingly, the number of INIs formed by A17-PABPN1 was reduced in myotubes treated with 17-AAG, suggesting that mutant A17-PABPN1 is degraded by the 17-AAG treatment. To obtain additional evidence to support this hypothesis, we examined aggregate formation in A17-PABPN1-transfected HeLa cells in response to 17-AAG treatment alone. As shown in Fig 4C and 4D, A17-PABPN1-transfected HeLa cells that form large, visible aggregates in response to DMSO showed a reduction in the size and intensity of these aggregates in response to 17-AAG treatment (2 μM for 24 hr). To determine whether treatment with 17-AAG protects against PABPN1 toxicity, we transfected C2C12 cells with wild-type A10-PABPN1 and mutant A17-PABPN1 and studied the effects on cell viability. We found that 17-AAG (100 nM, 48 hr) significantly protected C2C12 cells against mutant A17-PABPN1-induced cell death (Fig 4E).

Bottom Line: Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs).Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo.The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP).

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China.

ABSTRACT

Background: Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear.

Results: In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells.

Conclusions: Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD.

No MeSH data available.


Related in: MedlinePlus