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Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake.

Ahn KH, Kim SK, Choi JM, Jung SY, Won JH, Back MJ, Fu Z, Jang JM, Ha HC, Kim DK - PLoS ONE (2013)

Bottom Line: Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells.Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase.Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, Dongjak-Ku, Seoul, South Korea.

ABSTRACT
Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH-optimum and Mg(2+)-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

No MeSH data available.


Related in: MedlinePlus

Effects of cations and PS on N-SMase activity of anti-Hsp60 antibody immunoprecipitates.The active fraction from Superose 12 gel filtration column was mixed with the specific antibody for Hsp60. The immunoprecipitated pellets were washed and aliquots of the immunoprecipitates were assayed for N-SMase activity. N-SMase activity was determined in the presence of different concentrations of Mg2+ and Mn2+ (A). N-SMase activity was determined in the presence of different concentrations of Ca2+ with or without 1 mM Mg2+ (B). The immunoprecipitates with the specific antibody for Hsp60 and N-SMase2 were pre-incubated with 50 µM PS for 10 min at 37°C, and then activity was measured with the standard assay system (C). These results represent mean ± S.D. from a single experiment performed in triplicate. Similar results were obtained in five independent experiments.
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pone-0067216-g005: Effects of cations and PS on N-SMase activity of anti-Hsp60 antibody immunoprecipitates.The active fraction from Superose 12 gel filtration column was mixed with the specific antibody for Hsp60. The immunoprecipitated pellets were washed and aliquots of the immunoprecipitates were assayed for N-SMase activity. N-SMase activity was determined in the presence of different concentrations of Mg2+ and Mn2+ (A). N-SMase activity was determined in the presence of different concentrations of Ca2+ with or without 1 mM Mg2+ (B). The immunoprecipitates with the specific antibody for Hsp60 and N-SMase2 were pre-incubated with 50 µM PS for 10 min at 37°C, and then activity was measured with the standard assay system (C). These results represent mean ± S.D. from a single experiment performed in triplicate. Similar results were obtained in five independent experiments.

Mentions: In order to confirm whether Hsp60 interact with N-SMase2, the pellets immunoprecipitated with anti-Hsp60 antibody were used for characterization of N-SMase. The N-SMase activity of the immunoprecipitates was dependent upon Mg2+ and Mn2+ (Figure 5A), whereas Cu2+, Zn2+ and Ni2+ were unable to stimulate activity (data not shown). The N-SMase activity of the immunoprecipitates was not activated by Ca2+ cation in the presence of 1 mM Mg2+, but it could be activated by 10−6 and 10−7 M Ca2+ in the absence of Mg2+ (Figure 5B). Interestingly, the N-SMase activity in the immunoprecipitates was activated by PS. Moreover, the IP study using N-SMase2 antibody revealed that the N-SMase activity of the immunoprecipitates was similar to the activity of immunoprecipitates by anti-Hsp60 antibody (Figure 5C). These results strongly suggest Hsp60 can interact with N-SMase2.


Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake.

Ahn KH, Kim SK, Choi JM, Jung SY, Won JH, Back MJ, Fu Z, Jang JM, Ha HC, Kim DK - PLoS ONE (2013)

Effects of cations and PS on N-SMase activity of anti-Hsp60 antibody immunoprecipitates.The active fraction from Superose 12 gel filtration column was mixed with the specific antibody for Hsp60. The immunoprecipitated pellets were washed and aliquots of the immunoprecipitates were assayed for N-SMase activity. N-SMase activity was determined in the presence of different concentrations of Mg2+ and Mn2+ (A). N-SMase activity was determined in the presence of different concentrations of Ca2+ with or without 1 mM Mg2+ (B). The immunoprecipitates with the specific antibody for Hsp60 and N-SMase2 were pre-incubated with 50 µM PS for 10 min at 37°C, and then activity was measured with the standard assay system (C). These results represent mean ± S.D. from a single experiment performed in triplicate. Similar results were obtained in five independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3686747&req=5

pone-0067216-g005: Effects of cations and PS on N-SMase activity of anti-Hsp60 antibody immunoprecipitates.The active fraction from Superose 12 gel filtration column was mixed with the specific antibody for Hsp60. The immunoprecipitated pellets were washed and aliquots of the immunoprecipitates were assayed for N-SMase activity. N-SMase activity was determined in the presence of different concentrations of Mg2+ and Mn2+ (A). N-SMase activity was determined in the presence of different concentrations of Ca2+ with or without 1 mM Mg2+ (B). The immunoprecipitates with the specific antibody for Hsp60 and N-SMase2 were pre-incubated with 50 µM PS for 10 min at 37°C, and then activity was measured with the standard assay system (C). These results represent mean ± S.D. from a single experiment performed in triplicate. Similar results were obtained in five independent experiments.
Mentions: In order to confirm whether Hsp60 interact with N-SMase2, the pellets immunoprecipitated with anti-Hsp60 antibody were used for characterization of N-SMase. The N-SMase activity of the immunoprecipitates was dependent upon Mg2+ and Mn2+ (Figure 5A), whereas Cu2+, Zn2+ and Ni2+ were unable to stimulate activity (data not shown). The N-SMase activity of the immunoprecipitates was not activated by Ca2+ cation in the presence of 1 mM Mg2+, but it could be activated by 10−6 and 10−7 M Ca2+ in the absence of Mg2+ (Figure 5B). Interestingly, the N-SMase activity in the immunoprecipitates was activated by PS. Moreover, the IP study using N-SMase2 antibody revealed that the N-SMase activity of the immunoprecipitates was similar to the activity of immunoprecipitates by anti-Hsp60 antibody (Figure 5C). These results strongly suggest Hsp60 can interact with N-SMase2.

Bottom Line: Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells.Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase.Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, Dongjak-Ku, Seoul, South Korea.

ABSTRACT
Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH-optimum and Mg(2+)-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.

No MeSH data available.


Related in: MedlinePlus