Selective mutagenesis of lysyl residues leads to a stable and active form of delta 9 stearoyl-CoA desaturase.
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.
Mziaut
Hassan
H
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030-3305, USA.
Korza
George
G
Benraiss
Abdellatif
A
Ozols
Juris
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D017931:DNA Primers; D004795:Enzyme Stability; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics*; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013230:Stearoyl-CoA Desaturase; D025801:Ubiquitin / Q000378:metabolism
D017931:DNA Primers; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D025801:Ubiquitin; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine