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Studies of interactions between leukotriene C4 synthase, five-lipoxygenase activating protein and 5-lipoxygenase
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cysteinyl leukotrienes (cysLTs) are biologically active lipid mediators of great importance in asthma and inflammation. Three proteins are required to convert arachidonic acid into leukotriene C4 namely: five-lipoxygenase (5-LO), five-lipoxygenase activating protein (FLAP) and leukotriene C4 synthase (LTC4S). LTC4S and FLAP belong to the MAPEG (membrane associated proteins in eicosanoid and glutathione metabolism) family of proteins and are located on the nuclear envelope. Upon cell activation 5-LO translocates from the cytosol to the nuclear envelope enabling protein-protein interactions to occur between the three biosynthetic enzymes. GST pull-down experiments in this study demonstrate interaction between LTC4S and 5-LO, LTC4S and FLAP and between FLAP and 5-LO. Experiments with truncated mutants indicated that the second hydrophilic loop of LTC4S is important for interaction with 5-LO, and that the N-terminal part of LTC4S is important for FLAP interaction. Bioluminescence resonance energy transfer (BRET) experiments in transfected cells provided additional evidence that LTC4S interacts with 5-LO.

National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-81998OAI: oai:DiVA.org:liu-81998DiVA: diva2:557405
Available from: 2012-09-27 Created: 2012-09-27 Last updated: 2013-10-23
In thesis
1. Leukotriene C4 synthase: studies on oligomerization and subcellular localization
Open this publication in new window or tab >>Leukotriene C4 synthase: studies on oligomerization and subcellular localization
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Leukotrienes (LTs) are polyunsaturated fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 can be hydrolyzed to LTB4, or be conjugated with glutathione forming LTC4. LTC4 together with its metabolites LTD4 and LTE4, formed by amino acid removal from the glutathione moiety, constitute the cysteinyl LTs that are the active compounds of "slow reacting substance of anaphylaxis" (SRS-A). LTC4 and LTD4 are involved in several inflammatory conditions, e.g. asthma and allergic rhinitis. The conversion of LTA4 to LTC4 is catalyzed by an integral membrane protein, LTC4 synthase (LTC4S), localized on the endoplasmic reticulum (ER) and nuclear envelope. This 150 amino acid protein has four transmembrane helices and two hydrophilic loops oriented to the lumen side of the ER membrane. LTC4S belongs to a family of proteins called membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG).

We have shown that LTC4S and another MAPEG member, microsomal glutathione S-transferase (MGST)-1, interact and colocalize in transiently transfected cells. Coexpression decreased their catalytic activities indicating functional significance of the interaction between LTC4S and MGST1. LTC4S was demonstrated to form homo-oligomers in cell free assays using GST pulldown assays, as well as in living cells using bioluminescence resonance energy transfer (BRET) technique. When testing various truncated variants of LTC4S in BRET assays two hydrophobic regions were mapped as interaction domains: amino acids 6-27 and 114-135. GFP-fusion proteins containing the latter sequence also showed distinct ER/nuclear envelope localization and a minimal ER/nuclear envelope localization sequence was mapped to amino acids 117-132. In cell free assays we also demonstrated interactions between 5-LO, fivelipoxygenase activating protein (FLAP) and LTC4S. The second hydrophilic loop of LTC4S was found to be important for interaction with 5-LO, whereas the N-terminal part of LTC4S gave the strongest interaction with FLAP. LTC4 diminished the interaction between 5-LO and FLAP suggesting a feed-back regulatory mechanism. Our results concerning LTC4S oligomer formation and mapping of interaction domains may provide novel means to rational design of LTC4S inhibitors.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2005. 73 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 913
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-31123 (URN)16857 (Local ID)91-85299-23-5 (ISBN)16857 (Archive number)16857 (OAI)
Public defence
2005-10-14, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2014-06-13Bibliographically approved

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Svartz, JesperHallin, ElisabethFranck, NiclasStrid, TobiasSöderström, Mats

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