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Studies of Membrane Topology of Mitochondrial Cholesterol Hydroxylases CYPs 27A1 and 11A1

  • Original Article
  • Published:
Lipids

Abstract

Mitochondrial cytochrome P450 enzymes (CYP or P450, EC 1.14.13.15) play an important role in metabolism of cholesterol. CYP27A1 hydroxylates cholesterol at position 27 and, thus, initiates cholesterol removal from many extrahepatic tissues. CYP11A1 is a steroidogenic P450 that converts cholesterol to pregnenolone, the first step in the biosynthesis of all steroid hormones. We utilized a new approach to study membrane topology of CYPs 27A1 and 11A1. This approach involves heterologous expression of membrane-bound P450 in E. coli, isolation of the P450-containing E. coli membranes, treatment of the membranes with protease, removal of the digested soluble portion and extraction of the membrane-associated peptides, which are then identified by mass spectrometry. By using this approach, we found four membrane-interacting peptides in CYP27A1, and two peptides in CYP11A1. Peptides in CYP27A1 represent a contiguous portion of the polypeptide chain (residues 210–251) corresponding to the putative F-G loop and adjacent portions of the F and G helices. Peptides in CYP11A1 are from the putative F-G loop (residues 218–225) and the C-terminal portion of the G helix (residues 238–250). This data is consistent with those obtained previously by us and others and provide new information about the membrane topology of CYPs 27A1 and 11A1.

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Abbreviations

CYP27A1:

Cytochrome P450 27A1

CYP11A1:

Cytochrome P450 11A1

MS:

Mass spectrometry

TFA:

Trifluoroacetic acid

MALDI:

MS matrix assisted laser desorption ionization mass spectrometry

TOF:

Time-of-flight

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Acknowledgments

This research was supported in part by the grants from the National Institutes of Health GM062882 and AG024336 (to I.A.P.). Certain commercial materials, instruments, and equipment are identified in this manuscript in order to specify the experimental procedure as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology (NIST) nor does it imply that the materials, instruments, or equipment identified is necessarily the best available for the purpose

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Author notes

  1. Irina A. Pikuleva & Natalia Mast

    Present address: Department of Ophthalmology and Visual Sciences, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, OH, 44106, USA

Authors and Affiliations

  1. Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, 77555-1031, USA

    Irina A. Pikuleva & Natalia Mast

  2. Center for Advanced Research in Biotechnology, National Institute of Standards and Technology and University of Maryland Biotechnology Institute, Rockville, MD, 20850, USA

    Wei-Li Liao & Illarion V. Turko

Authors
  1. Irina A. Pikuleva
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  2. Natalia Mast
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  3. Wei-Li Liao
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  4. Illarion V. Turko
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Correspondence to Irina A. Pikuleva.

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Pikuleva, I.A., Mast, N., Liao, WL. et al. Studies of Membrane Topology of Mitochondrial Cholesterol Hydroxylases CYPs 27A1 and 11A1. Lipids 43, 1127–1132 (2008). https://doi.org/10.1007/s11745-008-3234-x

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  • DOI: https://doi.org/10.1007/s11745-008-3234-x

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