Expression, purification, and biochemical characterization of SAG, a ring finger redox-sensitive protein.
We recently reported the cloning and characterization of SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, that is redox responsive and protects mammalian cells from apoptosis. Here we report the expression, purification, and biochemical characterization of SAG. Bacterially expressed SAG is brown in color and dithiothreitol (DTT)-sensitive. SAG forms large oligomers without DTT that can be reduced into a monomer in the presence of DTT. These features help us to purify SAG using the chromatography with or without DTT. Likewise, purified SAG is redox sensitive. Upon H2O2 exposure, SAG forms oligomers as well as monomer doublets due to the formation of the inter- or intramolecular disulfide bonds, respectively. This process can be reversed by DTT or prevented by pretreatment with the alkylating reagent, N-ethylmaleimide (NEM). Although SAG contains two putative heme-binding sites and a RING finger domain, the protein appears not to bind with heme and to lack transcription factor activity as determined in a Gal4-fusion/transactivation assay. Wildtype, but not RING finger domain-disrupted SAG mutants, prevents copper-induced lipid peroxidation. These results, along with our previous observations, suggest that SAG is an intracellular antioxidant molecule that may act as a redox sensor to buffer oxidative-stress induced damage.
Swaroop
M
M
Department of Molecular Biology, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA.
Bian
J
J
Aviram
M
M
Duan
H
H
Bisgaier
C L
CL
Loo
J A
JA
Sun
Y
Y
D003300:Copper; D004926:Escherichia coli; D016166:Free Radical Scavengers / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006418:Heme / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D010084:Oxidation-Reduction; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases; D016335:Zinc Fingers
D016166:Free Radical Scavengers; D008077:Lipoproteins, LDL; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D006418:Heme; D003300:Copper; D007501:Iron; C118307:RNF7 protein, human; D044767:Ubiquitin-Protein Ligases