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 Mg2+-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.

Roles and regulation of neutral sphingomyelinase-2 in cellular and pathological processes

“…Effects of nSMase2 on normal synaptic regulation was studied in models of dopamine uptake where it was shown that nSMase2 inhibition or downregulation by siRNA decreased dopamine uptake in PC12 cells (Kim et al , 2010) with Hsp60 acting as an interaction partner that negatively regulates nSMase2 in that system (Ahn et al , 2013). This paved the way to more in depth studies of involvement of nSMase2 in dopaminergic pathologies such as Parkinson’s disease.…”

Purification and Characterization of Mitochondrial Mg2+-Independent Sphingomyelinase from Rat Brain

“…3A , lower panel). A previous study reported that nSMase2 directly interacts with Hsp60 via negative regulation ( Ahn et al, 2013 ); hence, we examined whether mt-iSMase binds to Hsp60. Mitochondrial and synaptosomal fractions were immunoprecipitated using anti-Hsp60 and anti-c-Myc antibodies ( Fig.…”

Inhibition of neutral sphingomyelinase protects mice against systemic tuberculosis

“…Additional studies have shown that Nsm is also regulated by heat shock protein 60 and by the phosphatase calcineurin (44,45). Although calcineurin has been previously shown to be involved in mycobacterial infections, it is presently unknown whether calcineurin also plays a role in mycobacteria-induced activation of Nsm (46).…”