کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1926475 | 1536461 | 2009 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Combined use of mass spectrometry and heterologous expression for identification of membrane-interacting peptides in cytochrome P450 46A1 and NADPH-cytochrome P450 oxidoreductase
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solution-exposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Archives of Biochemistry and Biophysics - Volume 483, Issue 1, 1 March 2009, Pages 81-89
Journal: Archives of Biochemistry and Biophysics - Volume 483, Issue 1, 1 March 2009, Pages 81-89
نویسندگان
Natalia Mast, Wei-Li Liao, Irina A. Pikuleva, Illarion V. Turko,