title: Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake.
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.
authors.affiliation: Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, Dongjak-Ku, Seoul, South Korea.
authors.forename: Kyong-Hoon
authors.initials: KH
authors.lastname: Ahn
authors.forename: Seok-Kyun
authors.initials: SK
authors.lastname: Kim
authors.forename: Jong-Min
authors.initials: JM
authors.lastname: Choi
authors.forename: Sung-Yun
authors.initials: SY
authors.lastname: Jung
authors.forename: Jong-Hoon
authors.initials: JH
authors.lastname: Won
authors.forename: Moon-Jung
authors.initials: MJ
authors.lastname: Back
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Fu
authors.forename: Ji-Min
authors.initials: JM
authors.lastname: Jang
authors.forename: Hae-Chan
authors.initials: HC
authors.lastname: Ha
authors.forename: Dae-Kyong
authors.initials: DK
authors.lastname: Kim
mesh_terms: D000818:Animals; D002417:Cattle; D018834:Chaperonin 60 / Q000502:physiology*; D004298:Dopamine / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016716:PC12 Cells; D051381:Rats; D017207:Rats, Sprague-Dawley; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*
chemical_list: D018834:Chaperonin 60; D013108:Sphingomyelin Phosphodiesterase; D004298:Dopamine