Macromolecular Machines in Leukocyte Function and Inflammation
- The Leukotriene Synthetic Complex. The leukotrienes (LTs) are bioactive signaling molecules generated in myeloid cells from arachidonic acid (AA) that initiate and amplify innate and adaptive immunity. There are two parent LTs, LTB? and LTC?. LTB? initiates and amplifies the innate immune response by functioning as a highly potent chemoattractant for PMNs, monocytes, mast cells, and certain T-cell subsets. LTC? and its products LTD? and LTE? have a variety of effects on airway and vascular smooth muscle. To prevent the unwanted initiation of inflammation and ensure that an inflammatory response is appropriately matched to a given challenge, cells have evolved a complex series of molecular controls to regulate LT synthesis. In resting cells, 5-lipoxygenase (5-LO), the initial enzyme of the LT pathway, resides in the cytosol and nucleoplasm, and cytosolic phospholipase A? (cPLA?) is localized in the cytosol. The substrate AA is esterified to phospholipids, and the generation of free AA by cPLA? is required for the oxygenation of AA by 5-LO. To initiate LT synthesis, increased intracellular calcium triggers the translocation of cPLA? to the endoplasmic reticulum (ER), outer nuclear membrane, and the Golgi. In parallel, 5-LO translocates to the inner and outer nuclear membranes. The N-terminal C2 domain of cPLA? and C2-like domain of 5-LO mediate their association with specific membrane phospholipids. On nuclear membranes, 5-LO must interact with its integral membrane scaffold protein, the 5-lipoxygenase-activating protein (FLAP). FLAP then functions to bring AA into apposition with 5-LO allowing the formation of the initial LT, LTA?. 5-LO and FLAP form the core of a multiprotein LT synthetic complex. The cores, along with other proteins, are further combined to form supramolecular nanoclusters.
As for many signaling complexes, the transient assembly of the LT synthetic complex on the nuclear membrane is governed by weak interactions between its members. These properties allow the LT synthetic complex to integrate multiple signals to achieve a graded output of LTs and to maintain biological flexibility; however, they also complicate analysis of composition and function. Different combinations of input signals can lead to different complex compositions, post-translational modifications of its members, and supramolecular organization. We are combining molecular imaging, cell biology, and biochemistry to define properties of this structure, identities of novel interacting proteins such as AP-10, and the supramolecular organization of LT synthesis.
- CD200R1 Signaling and Control of Viral Replication. Cells of the innate and adaptive immune systems contain a variety of transmembrane surface receptors that initiate or inhibit pro-inflammatory signaling. Inhibitory receptors dampen the amplitude and limit the duration of the inflammatory response. These receptors are activated by interaction with their ligands, which are also, commonly, cell surface proteins. We study CD200R1, a 301 amino acid transmembrane glycoprotein expressed on the surface of myeloid and glial cells. CD200R1 interacts with CD200, which is expressed on the surface of neurons, epithelial cells, endothelial cells, and lymphocytes. CD200R1 has a 67 amino acid intracellular domain with a unique inhibitory tyrosine motif. Using CD200R1 KO mice generated by our laboratory, we have found that Herpes Simplex Virus-1 (HSV-1) requires CD200R1 for successful viral infection of the brain, either via support of viral replication or by suppression of host defenses.
We have identified a surprising pro-inflammatory role for CD200R1 in the absence of CD200. In CD200R1 KO macrophages, the generation of IL-6 and CCL5 (Rantes) in response to ligands for TLR2 (but not TLR4) was blunted by 80% compared to WT cells. We are studying: the signaling pathways that interface with CD200R1, the protein interactions of CD200R1 in the presence, and absence of interaction with CD200 and its different cellular functions. The spatial and temporal regulation of signaling by CD200R1 in cells and in tissues is also being investigated. Finally, the mechanisms of how different viruses exploit different inhibitory receptor ligand pairs is under investigation.
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