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Title page for ETD etd-10182005-094007


Type of Document Dissertation
Author Yu, Zheyong
Author's Email Address zheyong.yu@vanderbilt.edu
URN etd-10182005-094007
Title Discovery of a novel lipoxygenase pathway in skin
Degree PhD
Department Pharmacology
Advisory Committee
Advisor Name Title
Jason D. Morrow Committee Chair
Alan R. Brash Committee Member
Diane S. Keeney Committee Member
H. Alex Brown Committee Member
Richard M. Breyer Committee Member
Keywords
  • lipoxygenase
  • ichthyosis
  • skin
  • epoxyalcohol
  • hepoxilin
  • PPAR
Date of Defense 2005-10-11
Availability unrestricted
Abstract
Lipoxygenase (LOX) are non-heme iron dioxygenases that form fatty acid hydroperoxides used in membrane remodeling and cell signaling. Mammalian epidermal LOX type 3 (eLOX3) is distinctive in totally lacking this typical oxygenase activity. Surprisingly, genetic evidence has linked mutations in either eLOX3 or a co-localizing enzyme, 12R-LOX, to an inherited skin disease, non-bullous congenital ichthyosiform erythroderma (NCIE), in which there is a defect in the normal skin permeability barrier (Hum. Mol. Gen. 11, 107-113). Here I identify a logical link of the biochemistry to the genetics. eLOX3 functions as a hydroperoxide isomerase utilizing the product of 12R-LOX, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE), as the preferred substrate. Using HPLC, GC-MS, NMR and CD spectroscopy, I demonstrated that eLOX3 converts 12R-HPETE to a specific epoxyalcohol, 8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid, and 12-ketoeicosatetraenoic acid in a 2:1 ratio. eLOX3 appears to be unique among LOX enzymes in using the ferrous form of the catalytic iron as the active species, initiating reaction by a one electron reduction of the substrate hydroperoxide and completing reaction by rebound hydroxylation to form the epoxyalcohol product. I analyzed the effect of the naturally occurring mutations identified in NCIE on eLOX3 and 12R-LOX catalytic activity; the lipoxygenase activity of 12R-LOX and the hydroperoxide isomerase activity of eLOX3 were totally eliminated. I further demonstrated that the epoxyalcohol formed by human eLOX3 is metabolized by soluble epoxide hydrolase in human keratinocytes to a single trihydroxy isomer, 8R,11S,12R-trihydroxyeicosa-5Z,9E,14Z-trienoic acid. Both the epoxyalcohol and its triol hydrolysis product were then tested for activity in activation of peroxisome proliferator-activated receptors (PPARs). Each selectively caused induction of PPARalpha-dependent transcription with similar activity to 8S-hydroxyeicosatetraenoic acid, a PPARalpha specific agonist. Because human and mouse express a different spectrum of LOX enzymes in skin, I also investigated the substrate selectivity of mouse eLOX3. It uses the product of mouse 8-LOX as its preferred substrate, a coupling consistent with the specific expression of 8-LOX in mouse skin. My results provide strong biochemical evidence for the existence of a novel LOX pathway. Loss of this pathway may contribute to a reduced differentiation in keratinocytes and pathogenesis of NCIE.
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