TY - JOUR
T1 - Neutral sphingomyelinase-2 is a redox sensitive enzyme
T2 - Role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state
AU - Dotson, P. Patrick
AU - Karakashian, Alexander A.
AU - Nikolova-Karakashian, Mariana N.
N1 - Publisher Copyright:
© The Authors Journal compilation © 2015 Biochemical Society.
PY - 2015/2/1
Y1 - 2015/2/1
N2 - Neutral sphingomyelinase-2 (nSMase-2) is themajor sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys617 to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state.
AB - Neutral sphingomyelinase-2 (nSMase-2) is themajor sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys617 to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state.
KW - Ceramide
KW - Cysteine
KW - Oligomerization
KW - Oxidative stress
KW - Sphingomyelinase
KW - Thioredoxin
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U2 - 10.1042/BJ20140665
DO - 10.1042/BJ20140665
M3 - Article
C2 - 25287744
AN - SCOPUS:84921646331
SN - 0264-6021
VL - 465
SP - 371
EP - 382
JO - Biochemical Journal
JF - Biochemical Journal
ER -