Leukotriene C4 synthase : studies on oligomerization and subcellular localization

Sammanfattning: 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.