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Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LOin vitro., QC 20200908

Inflammation is one of the innate defence mechanisms exerted by the human body for protection and to initiate the healing process. Chronic inflammatory reactions can lead to several disease conditions like asthma, allergic rhinitis and rheumatoid arthritis. A central role in these diseases is played by the Leukotriene (LT) family of the lipid mediators, one of family of pro-inflammatory lipid mediators derived from the 20-carbon fatty acid arachidonic acid. The key enzyme involved in LT biosynthesis is 5-Lipoxygenase (5LO) which initiates the leukotriene biosynthesis by catalysing the conversion of arachidonic acid (AA) to leukotriene A4 (LTA4). In this thesis, one aim is to elucidate the interactions of 5LO with the membrane and components therein which is the first step in the leukotriene biosynthesis and which could be one possible drug target. Furthermore, details of the LTA4 formation were investigated as well as LTA4 hydrolysis. In Paper I & II, we demonstrated the calcium-mediated binding of 5LO with membranes in vitro using a membrane substitute called nanodisc. The selected nanodisc platform contained 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and the membrane scaffolding protein MSP1E3D1 which provides a bilayer surface of inner diameter around 10 nm. Paper I shows 5LO binds directly with nanodiscs in the presence of calcium. This calcium-mediated binding was shown by non-denaturing gel-electrophoresis. Due to the high binding affinity, the 5LO-nanodisc complex was visible in the gel in the absence of calcium ions during the gel run. In the presence of Ca2+, a titration varying the ratio of 5LO to nanodiscs showed that maximum two 5LO binds to the nanodiscs used. The binding of 5LO with nanodiscs in the presence of calcium aides in the product formation and the results are comparable with the similar membrane mimic, liposomes. The complex of 5LO with nanodiscs was visualized for the first time by non-denaturing gel-electrophoresis and negative stain

QC 20130507