Limits...
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

Immunoprecipitation of N-SMase with antibody against Hsp60.(A) The active N-SMase fraction from Mono S was used for IP. The active fraction was mixed with the specific antibody for Hsp60. Aliquots from the immunoprecipitated supernatants were assayed for N-SMase activity. (B) The supernatant and immunoprecipitates were separated in 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with anti-Hsp60 antibody. (C) The active fraction was mixed with indicated concentrations of Triton X-100, and the respective specific antibodies for Hsp60 and N-SMase2 were added. The immunoprecipitated pellets were washed and aliquots of the immunoprecipitates were separated in a 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and probed with anti-Hsp60 and anti-N-SMase2 antibodies.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3686747&req=5

pone-0067216-g004: Immunoprecipitation of N-SMase with antibody against Hsp60.(A) The active N-SMase fraction from Mono S was used for IP. The active fraction was mixed with the specific antibody for Hsp60. Aliquots from the immunoprecipitated supernatants were assayed for N-SMase activity. (B) The supernatant and immunoprecipitates were separated in 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with anti-Hsp60 antibody. (C) The active fraction was mixed with indicated concentrations of Triton X-100, and the respective specific antibodies for Hsp60 and N-SMase2 were added. The immunoprecipitated pellets were washed and aliquots of the immunoprecipitates were separated in a 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and probed with anti-Hsp60 and anti-N-SMase2 antibodies.

Mentions: In order to investigate the possibility of an interaction between Hsp60 and N-SMase 2, co-IP studies were performed on purified fractions from bovine brain. Whereas incubation of the Mono S active fraction with increasing amounts of mouse Ig G2 (negative control) did not result in any loss of N-SMase activity, antibody against Hsp60 immunoprecipitated N-SMase activity in a dose-dependent manner (Fig 4A, B). As shown in Figure 4A, 4 µg of anti-Hsp60 Ab precipitated 20% of the N-SMase activity. Some 10% of total N-SMase activity could be recovered in the immunoprecipitates after resuspension in IP buffer (data not shown).


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)

Immunoprecipitation of N-SMase with antibody against Hsp60.(A) The active N-SMase fraction from Mono S was used for IP. The active fraction was mixed with the specific antibody for Hsp60. Aliquots from the immunoprecipitated supernatants were assayed for N-SMase activity. (B) The supernatant and immunoprecipitates were separated in 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with anti-Hsp60 antibody. (C) The active fraction was mixed with indicated concentrations of Triton X-100, and the respective specific antibodies for Hsp60 and N-SMase2 were added. The immunoprecipitated pellets were washed and aliquots of the immunoprecipitates were separated in a 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and probed with anti-Hsp60 and anti-N-SMase2 antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3686747&req=5

pone-0067216-g004: Immunoprecipitation of N-SMase with antibody against Hsp60.(A) The active N-SMase fraction from Mono S was used for IP. The active fraction was mixed with the specific antibody for Hsp60. Aliquots from the immunoprecipitated supernatants were assayed for N-SMase activity. (B) The supernatant and immunoprecipitates were separated in 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with anti-Hsp60 antibody. (C) The active fraction was mixed with indicated concentrations of Triton X-100, and the respective specific antibodies for Hsp60 and N-SMase2 were added. The immunoprecipitated pellets were washed and aliquots of the immunoprecipitates were separated in a 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and probed with anti-Hsp60 and anti-N-SMase2 antibodies.
Mentions: In order to investigate the possibility of an interaction between Hsp60 and N-SMase 2, co-IP studies were performed on purified fractions from bovine brain. Whereas incubation of the Mono S active fraction with increasing amounts of mouse Ig G2 (negative control) did not result in any loss of N-SMase activity, antibody against Hsp60 immunoprecipitated N-SMase activity in a dose-dependent manner (Fig 4A, B). As shown in Figure 4A, 4 µg of anti-Hsp60 Ab precipitated 20% of the N-SMase activity. Some 10% of total N-SMase activity could be recovered in the immunoprecipitates after resuspension in IP buffer (data not shown).

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