Selective mutagenesis of lysyl residues leads to a stable and active form of delta 9 stearoyl-CoA desaturase |
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Authors: | Hassan Mziaut George Korza Abdellatif Benraiss Juris Ozols |
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Institution: | 1. Department of Biochemistry, University of Connecticut Health Center, Farmington, CT 06030-3305, USA;2. Departments of Neurology and Neuroscience, Cornell University Medical College, New York, NY 10021, USA |
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Abstract: | Stearoyl-CoA desaturase (SCD) is a short-lived integral membrane protein of the endoplasmic reticulum (ER) that catalyzes the insertion of a double bond in the delta 9 position of saturated fatty acids. Its expression has been difficult in heterologous systems. In this study, recombinant adenovirus vector was used to express both wild-type (wt) and engineered forms of rat SCD in human transformed kidney cells. In the engineered form of SCD, lysyl residues at positions 33, 35, and 36 were mutated to alanine (SCD K/A). The recombinant adenovirus also contains a cDNA encoding the green fluorescent protein (GFP). The stable reporter GFP was used to analyze the efficiency of transfection and the stability of expressed SCDs. The wt SCD was unstable upon expression, whereas expression of SCD K/A resulted in the stabilization of the protein. The proteasome inhibitor MG132 did not affect the rapid degradation of expressed wt SCD, implying that proteasome is not involved in this degradation. Functional analysis of microsomes from infected cells expressing SCD K/A resulted in the formation of holoenzyme with desaturase activity. Here we report engineering a stabilized form of a rapidly degraded membrane protein for production of an active mutant form of SCD. The adenovirus transformed cells may provide a model for the study of the effects of positive SCD expression. |
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Keywords: | Desaturase Adenovirus Stabilized mutant Protein degradation AEBSF 4-(2-aminoethyl)benzenesulfonyl fluoride APP amyloid precursor protein DMSO dimethyl sulfoxide E-1 ubiquitin–activating enzyme E-64 ER endoplasmic reticulum FBS fetal bovine serum GFP green fluorescent protein HMGR 3-hydroxy-3-methylglutaryl coenzyme A reductase IPTG IRF-1 interferon regulatory factor MBP maltose binding protein MG132 SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SREBP sterol regulatory element-binding protein TLC thin layer chromatography |
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