Mast cells, macrophages, and polymorphonuclear leukocytes (PMN), initiate and amplify inflammation in the lung by synthesizing and releasing the bioactive lipids leukotriene (LT)B4, and LTC4. After its release from cells, LTC4 is converted to LTD4. These eicosanoids help """"""""shape"""""""" the pulmonary inflammatory response by regulating leukocyte recruitment, endothelial permeability, fibroblast proliferation, and smooth muscle contraction. LTC4, LTD4 and LTB4 are central to the pathogenesis of asthma and idiopathic pulmonary fibrosis (IFF). They are also critical for host defense to infection. How cells modulate the synthesis of these eicosanoids is therefore central to the pathogenesis of these diseases. The formation of LTC4 requires the functional interaction of at least four proteins on the nuclear envelope. These are cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), the 5-lipoxygenase-activating protein (FLAP), and LTC4 synthase;the metabolism of LTA by LTA4 hydrolase yields LTB4. We have used a combination of biochemistry and molecular imaging to demonstrate the interaction of 5-LO, FLAP, and LTC4 synthase in novel, activation- dependent, macromolecular complexes on the nuclear envelope that include additional proteins. We have also identified a 10 kDa protein (Associated Protein 10 kDa, AP-10) that dissociates from FLAP after cell activation concurrent with complex formation. The hypothesis of this proposal is that these complexes are the link between cell activation and LT synthesis. The broad long-term objective of this proposal is to understand how the assembly, degradation, and compartmentalization of these complexes regulate the formation of LTs on molecular, cell biological, and in vivo levels.
Three Specific Aims are proposed.
In Specific Aim 1 we will determine how 5-LO membrane complexes are assembled and organized.
In Specific Aim 2 we will identify the AP-10 protein.
In Specific Aim 3 we will test the hypothesis that the synthesis of LTC4 and LTB4 is compartmentalized between the cytosol and nucleus and that newly identified inner membrane 5-LO complexes regulate the synthesis of LTB4. The completion of these aims will allow the identification of novel mechanisms and, potentially, therapeutic approaches, to inflammatory pulmonary diseases.
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