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Expression and Characterization of Recombinant Neutral Sphingomyelinase 2 in Escherichia coli: Evidence of Sensitivity to Redox State
Author(s) -
Dotson P. Patrick,
NikolovaKarakashian Mariana
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1019.2
Subject(s) - sphingomyelin , cysteine , ceramide , biochemistry , chemistry , sphingomyelin phosphodiesterase , enzyme , microbiology and biotechnology , biology , cholesterol , apoptosis
Neutral sphingomyelinases are a family of lipid‐metabolizing enzymes that hydrolyze sphingomyelin to ceramide. Neutral sphingomyelinase 2 (nSMase‐2), in particular, was identified as the main cytokine‐inducible sphingomyelinase. Several recent studies have correlated nSMase‐2 activity with the state of oxidative stress. Depletion of cellular GSH, NADH oxidase, or exposure to agents that generate reactive oxygen species have been linked to upregulation of nSMase‐2 activity in various mammalian cells. The mechanisms for activation of nSMase‐2 during oxidative stress conditions, however, remain not completely understood. In this study, we investigate whether nSMase‐2 is a redox sensitive enzyme. A recombinant mouse nSMase‐2 construct was overexpressed in an Escherichia coli strain whereby recombinant proteins were produced that, based on in vitro activity assays, were functionally active. Interestingly, we demonstrate that nSMase‐2 exists in multimeric forms that are sensitive to reducing agents, which could correspond to cysteine‐mediated oligomerization between nSMase‐2 molecules. To directly test this, 17 of the 22‐ cysteine residues within nSMase‐2 were gradually mutated to serine. Gradual mutagenesis resulted in a loss of nSMase‐2 multimers with cysteine residues at position C561 and C617, within the catalytic domain, being identified as being involved in nSMase‐2 oligomerization. Furthermore, we show that the cysteine‐mediated nSMase‐2 oligomerization may be modulated through the thiol‐reducing enzyme thioredoxin. Together, these results suggest that nSMase‐2 is a redox‐sensitive enzyme and the redox state of specific cysteine residues may regulate nSMase‐2 activity.