Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2.

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple ß-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.

Cryo-EM structure of the human chemerin receptor 1-Gi protein complex bound to the C-terminal nonapeptide of chemerin.

Chemerin is a processed protein that acts on G protein-coupled receptors (GPCRs) for its chemotactic and adipokine activities. The biologically active chemerin (chemerin 21-157) results from proteolytic cleavage of prochemerin and uses its C-terminal peptide containing the sequence YFPGQFAFS for receptor activation. Here we report a high-resolution cryo-electron microscopy (cryo-EM) structure of human chemerin receptor 1 (CMKLR1) bound to the C-terminal nonapeptide of chemokine (C9) in complex with Gi proteins. C9 inserts its C terminus into the binding pocket and is stabilized through hydrophobic interactions involving its Y1, F2, F6, and F8, as well as polar interactions between G4, S9, and several amino acids lining the binding pocket of CMKLR1. Microsecond scale molecular dynamics simulations support a balanced force distribution across the whole ligand-receptor interface that enhances thermodynamic stability of the captured binding pose of C9. The C9 interaction with CMKLR1 is drastically different from chemokine recognition by chemokine receptors, which follow a two-site two-step model. In contrast, C9 takes an "S"-shaped pose in the binding pocket of CMKLR1 much like angiotensin II in the AT1 receptor. Our mutagenesis and functional analyses confirmed the cryo-EM structure and key residues in the binding pocket for these interactions. Our findings provide a structural basis for chemerin recognition by CMKLR1 for the established chemotactic and adipokine activities.

Agonist concentration-dependent changes in FPR1 conformation lead to biased signaling for selective activation of phagocyte functions.

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer-based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca2+ concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-ß/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca2+ mobilization from intracellular stores and Ca2+ influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca2+ level was required for degranulation and superoxide production but disrupted cytoplasmic Ca2+ concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and ß-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration-dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.

The 3-hydroxyacyl-CoA dehydratase 1/2 form complex with trans-2-enoyl-CoA reductase involved in substrates transfer in very long chain fatty acid elongation.

Very long-chain fatty acids (VLCFAs) are fatty acids with a carbon chain length greater than 18 carbons (>C18) and exhibit various functions, such as in skin barrier formation, liver homeostasis, myelin maintenance, spermatogenesis, retinal function, and anti-inflammation. VLCFAs are absorbed by dietary or elongated from endogenous hexadecanoyl acids (C16). Similar to long-chain fatty acid synthesis, VLCFAs elongation begins with acyl-CoA and malonyl-CoA as sources, and the length of the acyl chain is extended by two carbon units in each cycle. However, the VLCFAs elongation machinery is located in ER membrane and consists of four components, FA elongase (ELOVL), 3-ketoacyl-CoA reductase (KAR), 3-hydroxyacyl-CoA dehydratase (HACD), and trans-2-enoyl-CoA reductase (TECR), which is different with the long-chain fatty acid machinery fatty acid synthase (FAS) complex. Although the critical components in the elongation cycle are identified, the detailed catalytic and regulation mechanisms are still poorly understood. Here, we focused on the structural and biochemical analysis of TECR-associated VLCFA elongation reactions. Firstly, we identified a stable complex of human HACD2-TECR based on extensive in vitro characterizations. Combining computational modeling and biochemical analysis, we confirmed the critical interactions between TECR and HACD1/2. Then, we proposed the putative substrate binding sites and catalytic residues for TECR and HACD2. Besides, we revealed the structural similarities of HACD with ELOVLs and proposed the possible competition mechanism of TECR-associated complex formation.

Bidirectional regulation of neutrophil migration by mitogen-activated protein kinases.

To kill invading bacteria, neutrophils must interpret spatial cues, migrate and reach target sites. Although the initiation of chemotactic migration has been extensively studied, little is known about its termination. Here we found that two mitogen-activated protein kinases (MAPKs) had opposing roles in neutrophil trafficking. The extracellular signal-regulated kinase Erk potentiated activity of the G protein-coupled receptor kinase GRK2 and inhibited neutrophil migration, whereas the MAPK p38 acted as a noncanonical GRK that phosphorylated the formyl peptide receptor FPR1 and facilitated neutrophil migration by blocking GRK2 function. Therefore, the dynamic balance between Erk and p38 controlled neutrophil 'stop' and 'go' activity, which ensured that neutrophils reached their final destination as the first line of host defense.

Impaired p47phox phosphorylation in neutrophils from patients with p67phox-deficient chronic granulomatous disease.

Superoxide production by the phagocyte reduced NAD phosphate (NADPH) oxidase is essential for innate immunity as shown in chronic granulomatous disease (CGD), an immunodeficiency disease resulting from mutations in 1 of its genes. The NADPH oxidase is composed of 2 membrane proteins (gp91phox/NOX2 and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). The phosphorylation of p47phox is required for NADPH oxidase activation in cells. As p47phox and p67phox can form a tight complex in cells, we hypothesized that p67phox could regulate p47phox phosphorylation. To investigate this hypothesis, we used phospho-specific antibodies against 5 major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328, and Ser345) and neutrophils from healthy donors and from p67phox-/- CGD patients. Results showed that formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate induced a time- and a concentration-dependent phosphorylation of p47phox on Ser304, Ser315, Ser320, and Ser328 in healthy human neutrophils. Interestingly, in neutrophils and Epstein-Barr virus-transformed B lymphocytes from p67phox-/- CGD patients, phosphorylation of p47phox on serine residues was dramatically reduced. In COSphox cells, the presence of p67phox led to increased phosphorylation of p47phox. In vitro studies showed that recombinant p47phox was phosphorylated on Ser304, Ser315, Ser320, and Ser328 by different PKC isoforms and the addition of recombinant p67phox alone or in combination with p40phox potentiated this process. Thus, p67phox and p40phox are required for optimal p47phox phosphorylation on Ser304, Ser315, Ser320, and Ser328 in intact cells. Therefore, p67phox and p40phox are novel regulators of p47phox-phosphorylation.

The redox-sensitive cation channel TRPM2 modulates phagocyte ROS production and inflammation.

The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a nonselective and redox-sensitive cation channel, inhibited ROS production in phagocytic cells and prevented endotoxin-induced lung inflammation in mice. TRPM2-deficient mice challenged with endotoxin (lipopolysaccharide) had an enhanced inflammatory response and diminished survival relative to that of wild-type mice challenged with endotoxin. TRPM2 functioned by dampening NADPH oxidase-mediated ROS production through depolarization of the plasma membrane in phagocytes. As ROS also activate TRPM2, our findings establish a negative feedback mechanism for the inactivation of ROS production through inhibition of the membrane potential-sensitive NADPH oxidase.

Structural and conformational studies of biased agonism through formyl peptide receptors.

G protein-coupled chemoattractant receptors are class A GPCRs that couple primarily to the Gi class of heterotrimeric G proteins. Initially identified for their abilities to mediate leukocyte chemotaxis, chemoattractant GPCRs such as the formyl peptide receptors (FPRs) have been known for their diverse cellular functions in response to a variety of agonists. Stimulation of FPR2, in particular, leads to ligand-dependent activation of proinflammatory signaling as well as anti-inflammatory and proresolving signaling. Recently, the structures of FPR2-Gi protein complexed with ligands of different compositions have been solved by crystallization and cryo-electron microscopy. Analysis of the structural data as well as molecular simulation has led to the findings that the FPR2 binding pocket is sufficiently large for accommodation of several different types of ligands but in different poses. This mini-review focuses on the structural and conformational aspects of FPR2 for mechanisms underlying its biased agonism.

The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding.

Pathologic features of Alzheimer's disease (AD) include accumulation of amyloid ß (Aß) and hyperphosphorylated tau protein. We have shown previously that the chemokine-like receptor 1 (CMKLR1) is a functional receptor for Aß, and CMKLR1 contributes to the uptake of Aß. However, it is unclear whether CMKLR1 ameliorates or aggravates the process of AD. Here, we show that deletion of the gene coding for CMKLR1 significantly increased Aß deposits in brains of both male and female amyloid ß precursor protein/presenilin-1 mice. However, it markedly decreased the mortality of these mice. Behavioral studies found that CMKLR1 deficiency improved cognitive impairment of male and female amyloid ß precursor protein/presenilin-1 mice and intracerebroventricular-streptozotocin injection AD mice. We further explored the effect of CMKLR1 on tau pathology. We found that CMKLR1 deficiency or inhibition attenuated the hyperphosphorylation of tau in brains of AD mice in vivo and in the neuronal cells in vitro The expression of CMKLR1 on the neurons affected tau phosphorylation by participating in tau seeding. Together, these results uncover a novel mechanism of CMKLR1 in the pathologic process of AD and suggest that inhibiting the promotion effect of CMKLR1 on tau seeding may provide a new strategy for the treatment of AD.SIGNIFICANCE STATEMENT Evidence suggests that inflammation is involved in the pathologic progression of AD. The chemokine-like receptor 1 (CMKLR1), belonging to the family of GPCRs, is able to bind and uptake amyloid ß. We show here, for the first time, that, although CMKLR1 deficiency increased amyloid ß deposits in AD mice, it reduced the mortality and improved the cognitive deficits of AD mice. We furthermore show that CMKLR1 deficiency or inhibition attenuated tau hyperphosphorylation in brains of AD model mice in vivo and in neuronal cells in vitro Finally, we first discovered that the expression of CMKLR1 on neurons affected tau phosphorylation by participating in tau seeding. These findings suggest that inhibition of CMKLR1 may provide a new strategy for the treatment of AD.

Exploring the Activation Process of the ß2AR-Gs Complex.

G-Protein-coupled receptors (GPCRs) belong to an important family of integral membrane receptor proteins that are essential for a variety of transmembrane signaling process, such as vision, olfaction, and hormone responses. They are also involved in many human diseases (Alzheimer's, heart diseases, etc.) and are therefore common drug targets. Thus, understanding the details of the GPCR activation process is a task of major importance. Various types of crystal structures of GPCRs have been solved either at stable end-point states or at possible intermediate states. However, the detailed mechanism of the activation process is still poorly understood. For example, it is not completely clear when the nucleotide release from the G protein occurs and how the key residues on α5 contribute to the coupling process and further affect the binding specificity. In this work we show by free energy analysis that the guanosine diphosphate (GDP) molecule could be released from the Gs protein when the binding cavity is half open. This occurs during the transition to the Gs open state, which is the rate-determining step in the system conformational change. We also account for the experimentally observed slow-down effects by the change of the reaction barriers after mutations. Furthermore, we identify potential key residues on α5 and validated their significance by site-directed mutagenesis, which illustrates that computational works have predictive value even for complex biophysical systems. The methodology of the current work may be applied to other biophysical systems of interest.

Predicting Mutational Effects on Receptor Binding of the Spike Protein of SARS-CoV-2 Variants.

The pandemic caused by SARS-CoV-2 has cost millions of lives and tremendous social/financial loss. The virus continues to evolve and mutate. In particular, the recently emerged "UK", "South Africa", and Delta variants show higher infectivity and spreading speed. Thus, the relationship between the mutations of certain amino acids and the spreading speed of the virus is a problem of great importance. In this respect, understanding the mutational mechanism is crucial for surveillance and prediction of future mutations as well as antibody/vaccine development. In this work, we used a coarse-grained model (that was used previously in predicting the importance of mutations of N501) to calculate the free energy change of various types of single-site or combined-site mutations. This was done for the UK, South Africa, and Delta mutants. We investigated the underlying mechanisms of the binding affinity changes for mutations at different spike protein domains of SARS-CoV-2 and provided the energy basis for the resistance of the E484 mutant to the antibody m396. Other potential mutation sites were also predicted. Furthermore, the in silico predictions were assessed by functional experiments. The results establish that the faster spreading of recently observed mutants is strongly correlated with the binding-affinity enhancement between virus and human receptor as well as with the reduction of the binding to the m396 antibody. Significantly, the current approach offers a way to predict new variants and to assess the effectiveness of different antibodies toward such variants.

Dual modulation of formyl peptide receptor 2 by aspirin-triggered lipoxin contributes to its anti-inflammatory activity.

The eicosanoid lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 (ATL) are potent anti-inflammatory agents. How their anti-inflammatory effects are mediated by receptors such as the formyl peptide receptor 2 (FPR2/ALX) remains incompletely understood. In the present study, fluorescent biosensors of FPR2/ALX were prepared and ATL-induced conformational changes were recorded. A biphasic dose curve consisting of a descending phase and an ascending phase was observed, with the descending phase corresponding to diminished FPR2 response such as Ca2+ mobilization induced by the potent synthetic agonist WKYMVm. Preincubation of FPR2-expressing cells with 100 pM of ATL also lowered the threshold for WKYMVm to induce ß-arrestin-2 membrane translocation, and inhibited WKYMVm-induced interleukin 8 secretion, suggesting signaling bias favoring anti-inflammatory activities. At 100 pM and above, ATL-induced receptor conformational changes resembling that of the WKYMVm along with a weak but measurable inhibition of forskolin-induced cAMP accumulation. However, no Ca2+ mobilization was induced by ATL until its concentration reached 1 µM. Taken together, these results suggest a dual regulatory mechanism by which ATL exerts anti-inflammatory effects through FPR2/ALX.

The Kinesin Light Chain-Related Protein PAT1 Promotes Superoxide Anion Production in Human Phagocytes.

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function.

TGFß2-mediated epithelial-mesenchymal transition and NF-κB pathway activation contribute to osimertinib resistance.

Osimertinib (AZD9291) has been widely used for the treatment of EGFR mutant non-small cell lung cancer. However, resistance to osimertinib is inevitable. In this study we elucidated the molecular mechanisms of resistance in osimertinib-resistant NCI-H1975/OSIR cells. We showed that NCI-H1975/OSIR cells underwent epithelial-mesenchymal transition (EMT), which conferred sensitivity to the GPX4 inhibitor 1S, 3R-RSL3 to induce ferroptotic cell death. The EMT occurrence resulted from osimertinib-induced upregulation of TGFß2 that activated SMAD2. On the other hand, we revealed that NCI-H1975/OSIR cells were highly dependent on NF-κB pathway for survival, since treatment with the NF-κB pathway inhibitor BAY 11-7082 or genetic silence of p65 caused much greater cell death as compared with the parental NCI-H1975 cells. In NCI-H1975 cells, osimertinib activated NF-κB pathway, evidenced by the increased p65 nuclear translocation, which was abolished by knockdown of TGFß2. In the cancer genome atlas lung adenocarcinoma data, TGFB2 transcript abundance significantly correlated with EMT-associated genes and NF-κB pathway. In addition, coexistence of EMT and activation of NF-κB pathway was observed in several NCI-H1975/OSIR clones. These findings shed new light on distinct roles of TGFß2 in osimertinib-resistant cells and provide new strategies for treatment of this resistant status.

Serum amyloid A3 confers protection against acute lung injury in Pseudomonas aeruginosa-infected mice.

Pseudomonas aeruginosa is a gram-negative bacterium associated with serious illnesses, including ventilator-associated pneumonia and various sepsis syndromes in humans. Understanding the host immune mechanisms against P. aeruginosa is, therefore, of clinical importance. The present study identified serum amyloid A3 (SAA3) as being highly inducible in mouse bronchial epithelium following P. aeruginosa infection. Genetic deletion of Saa3 rendered mice more susceptible to P. aeruginosa infection with decreased neutrophil superoxide anion production, and ex vivo treatment of mouse neutrophils with recombinant SAA3 restored the ability of neutrophils to produce superoxide anions. The SAA3-deficient mice showed exacerbated inflammatory responses, which was characterized by pronounced neutrophil infiltration, elevated expression of TNF-α, KC/CXCL1, and MIP-2/CXCL2 in bronchoalveolar lavage fluid (BALF), and increased lung microvascular permeability compared with their wild-type littermates. BALF neutrophils from Saa3 knockout mice exhibited reduced superoxide anion production compared with neutrophils from wild-type mice. Adoptive transfer of SAA3-treated neutrophils to Saa3 knockout mice ameliorated P. aeruginosa-induced acute lung injury. These findings demonstrate that SAA3 not only serves as a biomarker for infection and inflammation, but also plays a protective role against P. aeruginosa infection-induced lung injury in part through augmentation of neutrophil bactericidal functions.

The Rho guanine nucleotide exchange factor P-Rex1 as a potential drug target for cancer metastasis and inflammatory diseases.

Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a guanine nucleotide exchange factor (GEF) for Rac small GTPases and the Rac-related GTPase RhoG. P-Rex1 plays an important role in cell migration and relays intracellular signals generated through activation of G protein-coupled receptors and receptor tyrosine kinases. Studies of mouse models have found that P-Rex1 expression and activation is associated with tumor cell migration, brain development and pathological changes such as lung edema. Since its initial discovery, P-Rex1 has been known for its large size and multiple activation mechanisms that involve not only PIP3 but also the ßγ subunits of heterotrimeric G proteins and a regulatory subunit of cyclic AMP-dependent kinase, PKA RIα. At the core of the GEF activity is the tandem Dbl homology domain and the pleckstrin homology domain (DH/PH domains) that are masked until activation signals unwind the P-Rex1 structure. Understanding the activation mechanisms will help designing therapeutics that target P-Rex1 for cancer and other diseases.

Biased allosteric modulation of formyl peptide receptor 2 leads to distinct receptor conformational states for pro- and anti-inflammatory signaling.

BACKGROUND AND PURPOSE: Formyl peptide receptor 2 (FPR2) is a Class A G protein-coupled receptor (GPCR) that interacts with multiple ligands and transduces both proinflammatory and anti-inflammatory signals. These ligands include weak agonists and modulators that are produced during inflammation. The present study investigates how prolonged exposure to FPR2 modulators influence receptor signaling. EXPERIMENTAL APPROACH: Fluorescent biosensors of FPR2 were constructed based on single-molecule fluorescent resonance energy transfer (FRET) and used for measurement of ligand-induced receptor conformational changes. These changes were combined with FPR2-mediated signaling events and used as parameters for the conformational states of FPR2. Ternary complex models were developed to interpret ligand concentration-dependent changes in FPR2 conformational states. KEY RESULTS: Incubation with Ac2-26, an anti-inflammatory ligand of FPR2, decreased FRET intensity at picomolar concentrations. In comparison, WKYMVm (W-pep) and Aß42, both proinflammatory agonists of FPR2, increased FRET intensity. Preincubation with Ac2-26 at 10 pM diminished W-pep-induced Ca2+ flux but potentiated W-pep-stimulated ß-arrestin2 membrane translocation and p38 MAPK phosphorylation. The opposite effects were observed with 10 pM of Aß42. Neither Ac2-26 nor Aß42 competed for W-pep binding at the picomolar concentrations. CONCLUSIONS AND IMPLICATIONS: The results support the presence of two allosteric binding sites on FPR2, each for Ac2-26 and Aß42, with high and low affinities. Sequential binding of the two allosteric ligands at increasing concentrations induce different conformational changes in FPR2, providing a novel mechanism by which biased allosteric modulators alter receptor conformations and generate pro- and anti-inflammatory signals.

Serum amyloid A promotes LPS clearance and suppresses LPS-induced inflammation and tissue injury.

Lipopolysaccharide (LPS) is a major microbial mediator for tissue injury and sepsis resulting from Gram-negative bacterial infection. LPS is an external factor that induces robust expression of serum amyloid A (SAA), a major constituent of the acute-phase proteins, but the relationship between SAA expression and LPS-induced tissue injury remains unclear. Here, we report that mice with inducible transgenic expression of human SAA1 are partially protected against inflammatory response and lung injury caused by LPS and cecal ligation and puncture (CLP). In comparison, transgenic SAA1 does not attenuate TNFα-induced lung inflammation and injury. The SAA1 expression level correlates inversely with the endotoxin concentrations in serum and lung tissues since SAA1 binds directly to LPS to form a complex that promotes LPS uptake by macrophages. Disruption of the SAA1-LPS interaction with a SAA1-derived peptide partially reduces the protective effect and exacerbates inflammation. These findings demonstrate that acute-phase SAA provides innate feedback protection against LPS-induced inflammation and tissue injury.

Anti-inflammatory signaling through G protein-coupled receptors.

G protein-coupled receptors (GPCRs) play important roles in human physiology. GPCRs are involved in immunoregulation including regulation of the inflammatory response. Chemotaxis of phagocytes and lymphocytes is mediated to a great extent by the GPCRs for chemoattractants including myriads of chemokines. Accumulation and activation of phagocytes at the site of inflammation contribute to local inflammatory response. A handful of GPCRs have been found to transduce anti-inflammatory signals that promote resolution of inflammation. These GPCRs interact with selected metabolites of arachdonic acid, such as lipoxins, and of omega-3 essential fatty acids, such as resolvins and protectins. Despite mounting evidence for the in vivo functions of these anti-inflammatory and pro-resolving ligands paired with their respective GPCRs, the underlying signaling mechanisms have not been fully delineated. The present review summarizes what we have learned about these GPCRs, their structures and signaling pathways and the prospect of targeting these receptors for novel anti-inflammatory therapies.

MLN4924 protects against interleukin-17A-induced pulmonary inflammation by disrupting ACT1-mediated signaling.

An excessive inflammatory response in terminal airways, alveoli, and the lung interstitium eventually leads to pulmonary hypertension and chronic obstructive pulmonary disease. Proinflammatory cytokine interleukin-17A (IL-17A) has been implicated in the pathogenesis of pulmonary inflammatory diseases. MLN4924, an inhibitor of NEDD8-activating enzyme (NAE), is associated with the treatment of various types of cancers, but its role in the IL-17A-mediated inflammatory response has not been identified. Here, we report that MLN4924 can markedly reduce the expression of proinflammatory cytokines and chemokines such as IL-1ß, IL-6, and CXCL-1 and neutrophilia in a mouse model of IL-17A adenovirus-induced pulmonary inflammation. MLN4924 significantly inhibited IL-17A-induced stabilization of mRNA of proinflammatory cytokines and chemokines in vitro. Mechanistically, MLN4924 significantly blocked the activation of MAPK and NF-κB pathways and interfered with the interaction between ACT1 and tumor necrosis factor receptor-associated factor proteins (TRAFs), thereby inhibiting TRAF6 ubiquitination. Taken together, our data uncover a previously uncharacterized inhibitory effect of MLN4924 on the IL-17A-mediated inflammatory response; this phenomenon may facilitate the development of MLN4924 into an effective small-molecule drug for the treatment of pulmonary inflammatory diseases.