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.
Highlights
Sphingolipids are known to be important components of signaling pathways because their metabolism gives rise to products that function as second messengers, such as ceramide, sphingosine, and sphingosine-1-phosphate
Since N-SMase2 is already known to be extracted with Triton X-100, we focused on salt-extracted neutral pH–optimum and Mg2+-dependent SMase (N-SMase), which may be a novel form in the membrane fractions of bovine brain
Based on our previous identification of multiple forms of neutral SMase (NSMase) from bovine brain [13], N-SMase can be classified into two groups according to the agent solubilizing the activity in the membrane fractions: (1) a Triton X-100-extractable N-SMase which further consists of four forms of N-SMase (a, b, c, and d) and (2) a salt-extractable N-SMase which further consists of two forms of N-SMase
Summary
Sphingolipids are known to be important components of signaling pathways because their metabolism gives rise to products that function as second messengers, such as ceramide, sphingosine, and sphingosine-1-phosphate. Ceramide, which is a central metabolite, is recognized as an important lipid messenger that plays key roles in a variety of cellular responses, including cell cycle arrest, apoptosis, senescence, and stress responses [1]. As a lipid second messenger, ceramide is generated in response to a variety of stimuli, including TNF-a [3], Fas ligand [4], phorbol ester [5], heat stress [6], oxidative stress [7], ionizing radiation [8], and chemotherapeutics [9]. By using a bioinformatics-based approach, 47.5kDa N-SMase1 [14], 71-kDa N-SMase2 [15], and 97-kDa NSMase3 [16] have been cloned and characterized
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