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Leukotriene C4 synthase: studies on oligomerization and subcellular localization
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-31123Local ID: 16857ISBN: 91-85299-23-5 (print)OAI: oai:DiVA.org:liu-31123DiVA: diva2:251946
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
List of papers
1. Colocalization of leukotriene C synthase and microsomal glutathione S-transferase elucidated by indirect immunofluorescence analysis
Open this publication in new window or tab >>Colocalization of leukotriene C synthase and microsomal glutathione S-transferase elucidated by indirect immunofluorescence analysis
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2000 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 480, no 2-3, 239-243 p.Article in journal (Refereed) Published
Abstract [en]

We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein–protein interaction may contribute to the regulation of LTC4 production.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-47589 (URN)10.1016/S0014-5793(00)01885-8 (DOI)000089209900035 ()
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
2. Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer
Open this publication in new window or tab >>Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer
2003 (English)In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1633, no 2, 90-95 p.Article in journal (Refereed) Published
Abstract [en]

Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC4 and its metabolites LTD4 and LTE4 stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC4 is formed by addition of glutathione to LTA4, catalyzed by the integral membrane protein, LTC4 synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114–150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57–88. The functional significance of LTCS homo-oligomer formation is currently being investigated.

Place, publisher, year, edition, pages
Elsevier, 2003
Keyword
eicosanoid; fusion protein; green fluorescent protein; human embryonic kidney cell; oligomerization
National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:liu:diva-25056 (URN)10.1016/S1388-1981(03)00091-X (DOI)000184499300003 ()9484 (Local ID)9484 (Archive number)9484 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
3. Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization
Open this publication in new window or tab >>Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization
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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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-36053 (URN)10.1002/jcb.20880 (DOI)000239469800013 ()29608 (Local ID)29608 (Archive number)29608 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
4. Studies of interactions between leukotriene C4 synthase, five-lipoxygenase activating protein and 5-lipoxygenase
Open this publication in new window or tab >>Studies of interactions between leukotriene C4 synthase, five-lipoxygenase activating protein and 5-lipoxygenase
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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:nbn:se:liu:diva-81998 (URN)
Available from: 2012-09-27 Created: 2012-09-27 Last updated: 2013-10-23

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