A 5‑lipoxygenase-specific sequence motif impedes enzyme activity and confers dependence on a partner protein

Schexnaydre, Erin; Gerstmeier, Jana; Garscha, Ulrike; Jordan, Paul M.; Werz, Oliver; Newcomer, Marcia E.

doi:10.1016/j.bbalip.2018.09.011

Cited by 4 publications

(8 citation statements)

References 39 publications

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“…EDTA is kept in our buffers which reduces trace metal availability for radical formation due to Fenton reactions. In fact, FLAP has been described as a gatekeeper or helper and it was proposed that the N-terminal tail of 5LO carrying a FY-motif acts like a cork and regulator [ 15 ]. In cells, non-bound 5LO is protected from non-turn-over inactivation by this cork and remains stable in the cells up to 4 hours with or without FLAP present in the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…In cells, non-bound 5LO is protected from non-turn-over inactivation by this cork and remains stable in the cells up to 4 hours with or without FLAP present in the membrane. However, upon recruitment to the membranes by Ca 2+ addition, 5LO was rapidly inactivated by a turn-over dependent mechanism [ 15 ]. It appeared that without or with Ca 2+ , 5LO is safe as long as FLAP is absent.…”

Section: Discussionmentioning

confidence: 99%

“…terminal tail of 5LO carrying a FY-motif acts like a cork and regulator [15]. In cells, nonbound 5LO is protected from non-turn-over inactivation by this cork and remains stable in the cells up to 4 hours with or without FLAP present in the membrane.…”

Section: Plos Onementioning

confidence: 99%

“…The formation of a productive LT producing protein complex was shown to be dependent on the presence of AA, endogenously provided by the release from the membrane storage due to the action of cPLA2, cytosolic phospholipase A2 [ 10 ]. A mutational analysis of 5LO showed that a triple lysine motif functions as a conformational switch regulator keeping the 5LO protected from turnover-based inactivation until a correct 5LO-FLAP interaction relieves the protection by the so called FY-cork for efficient LT synthesis [ 15 ]. This interaction between 5LO and FLAP was shown to depend on Cys159 in that a mutation of this cysteine to serine inhibited the 5LO-FLAP complex formation [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

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Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

et al. 2020

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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 Ca 2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca 2+-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 Ca 2 +-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 Ca 2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca 2+-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 5LO in vitro.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Plos Onementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

et al. 2020

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“…In fact, FLAP has been described as a gatekeeper or helper and it was proposed that the Nterminal tail of 5LO carrying a FY-motif acts like a cork and regulator (15). In cells, nonbound 5LO is protected from non-turn-over inactivation by this cork and remains stable in the cells up to 4 hours with or without FLAP present in the membrane.…”

Section: Either Ca 2+ or Aa Can Induce 5lofnd-complex Formation Stabmentioning

confidence: 99%

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

Kumar

Purhonen

Hebert

et al. 2020

Preprint

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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 Ca 2+ -ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca 2+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 Ca 2+ -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 spectroscopy. 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 Ca 2+ -flush is mainly needed for the recruitment of 5LO to the membrane.Our results also showed that the addition of Ca 2+ -ions promoted binding of 5LO on the FLAPnanodiscs 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 5LO in vitro.

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Structural and mechanistic insights into 5-lipoxygenase inhibition by natural products

et al. 2020

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Leukotrienes (LT) are lipid mediators of the inflammatory response that are linked to asthma and atherosclerosis. LT biosynthesis is initiated by 5-lipoxygenase (5-LOX) with the assistance of the substrate-binding 5-LOX-activating protein at the nuclear membrane. Here, we contrast the structural and functional consequences of the binding of two natural product inhibitors of 5-LOX. The redox-type inhibitor nordihydroguaiaretic acid (NDGA) is lodged in the 5-LOX active site, now fully exposed by disordering of the helix that caps it in the apo-enzyme. In contrast, the allosteric inhibitor 3-acetyl-11-keto-beta-boswellic acid (AKBA) from frankincense wedges between the membrane-binding and catalytic domains of 5-LOX, some 30 Å from the catalytic iron. While enzyme inhibition by NDGA is robust, AKBA promotes a shift in the regiospecificity, evident in human embryonic kidney 293 cells and in primary immune cells expressing 5-LOX.

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A 5‑lipoxygenase-specific sequence motif impedes enzyme activity and confers dependence on a partner protein

Cited by 4 publications

References 39 publications

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

Structural and mechanistic insights into 5-lipoxygenase inhibition by natural products

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