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Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization
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.ORCID iD: 0000-0003-3927-4394
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2006 (English)In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 98, no 6, 1517-1527 p.Article in journal (Refereed) Published
Abstract [en]

Leukotrienes (LTs) are 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 (NE) where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 is further converted to LTC4 by conjugation with glutathione, a reaction catalyzed by the integral membrane protein LTC4 synthase (LTC4S), which is localized on the NE and endoplasmic reticulum (ER). We now report the mapping of regions of LTC4S that are important for its subcellular localization. Multiple constructs encoding fusion proteins of green fluorescent protein (GFP) as the N-terminal part and various truncated variants of human LTC4S as C-terminal part were prepared and transfected into HEK 293/T or COS-7 cells. Constructs encoding hydrophobic region 1 of LTC4S (amino acids 6–27) did not give distinct membrane localized fluorescence. In contrast hydrophobic region 2 (amino acids 60–89) gave a localization pattern similar to that of full length LTC4S. Hydrophobic region 3 (amino acids 114–135) directed GFP to a localization indistinguishable from that of full length LTC4S. A minimal directing sequence, amino acids 117–132, was identified by further truncation. The involvement of the hydrophobic regions in the homo-oligomerization of LTC4S was investigated using bioluminescence resonance energy transfer (BRET) analysis in living cells. BRET data showed that hydrophobic regions 1 and 3 each allowed oligomerization to occur. These regions most likely form transmembrane helices, suggesting that homo-oligomerization of LTC4S is due to helix–helix interactions in the membrane.

Place, publisher, year, edition, pages
2006. Vol. 98, no 6, 1517-1527 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-36053DOI: 10.1002/jcb.20880ISI: 000239469800013Local ID: 29608OAI: oai:DiVA.org:liu-36053DiVA: diva2:256901
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
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)
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Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2014-06-13Bibliographically approved

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Svartz, JesperHallin, ElisabethShi, YixuanSöderström, MatsHammarström, Sven

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