title: Neutral sphingomyelinase-2 is a redox sensitive enzyme: role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state.
abstract: Neutral sphingomyelinase-2 (nSMase-2) is the major 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 Cys(617) 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.
authors.affiliation: *Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
authors.forename: P Patrick
authors.initials: PP
authors.lastname: Dotson
authors.affiliation: *Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Karakashian
authors.affiliation: *Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
authors.forename: Mariana N
authors.initials: MN
authors.lastname: Nikolova-Karakashian
mesh_terms: D000818:Animals; D002384:Catalysis / Q000187:drug effects; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D010084:Oxidation-Reduction / Q000187:drug effects; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000494:pharmacology
chemical_list: D013879:Thioredoxins; C491483:Smpd3 protein, mouse; D013108:Sphingomyelin Phosphodiesterase; D003545:Cysteine