Cytochrome b5 forms homomeric complexes in living cells
Autor: | Craig Adriaanse, Amanda C. Swart, Nicolaas Lombard, Pieter Swart, Karl-Heinz Storbeck |
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Rok vydání: | 2012 |
Předmět: |
Hemeprotein
Endocrinology Diabetes and Metabolism Molecular Sequence Data Clinical Biochemistry Biology Endoplasmic Reticulum environment and public health Biochemistry Endocrinology Chlorocebus aethiops Fluorescence Resonance Energy Transfer medicine Animals Homomeric Amino Acid Sequence Molecular Biology Sheep Domestic Base Sequence Cytochrome b Mutagenesis Steroid 17-alpha-Hydroxylase Cytochrome P450 Intracellular Membranes Cell Biology Lyase enzymes and coenzymes (carbohydrates) Cytochromes b5 CYP17A1 COS Cells embryonic structures Mutagenesis Site-Directed cardiovascular system Pregnenolone biology.protein Molecular Medicine Protein Multimerization Hydrophobic and Hydrophilic Interactions medicine.drug |
Zdroj: | The Journal of Steroid Biochemistry and Molecular Biology. 132:311-321 |
ISSN: | 0960-0760 |
Popis: | Cytochrome b(5) (cyt-b(5)) is a ubiquitous hemoprotein also associated with microsomal cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1). In the steroidogenic pathway CYP17A1 catalyses the metabolism of pregnenolone, yielding both glucocorticoid and androgen precursors. While not affecting the 17α-hydroxylation of pregnenolone, cyt-b(5) augments the 17,20 lyase reaction of 17-hydroxypregnenolone, catalyzing the formation of DHEA, through direct protein-protein interactions. In this study, multimeric complex formation of cyt-b(5) and the possible regulatory role of these complexes were investigated. Cyt-b(5) was isolated from ovine liver and used to raise anti-sheep cyt-b(5) immunoglobulins. Immunochemical studies revealed that, in vivo, cyt-b(5) is primarily found in the tetrameric form. Subsequent fluorescent resonance energy transfer (FRET) studies in COS-1 cells confirmed the formation of homomeric complexes by cyt-b(5) in live cells. Site-directed mutagenesis revealed that the C-terminal linker domain of cyt-b(5) is vital for complex formation. The 17,20-lyase activity of CYP17 was augmented by truncated cyt-b(5), which is unable to form complexes when co-expressed in COS-1 cells, thereby implicating the monomeric form of cyt-b(5) as the active species. This study has shown for the first time that cyt-b(5) forms homomeric complexes in vivo, implicating complex formation as a possible regulatory mechanism in steroidogenesis. |
Databáze: | OpenAIRE |
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