The omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) can suppress inflammation, specifically IL-1β production through poorly understood molecular mechanisms. Here, we show that DHA reduces macrophage IL-1β production by limiting inflammasome activation. Exposure to DHA reduced IL-1β production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR) 4 (also known as GPR120), a G-protein coupled receptor (GPR) known to bind DHA. The exposure of cells to DHA recruited the adapter protein β-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-κB. DHA also reduced IL-1β levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.

Autophagy is an essential eukaryotic pathway requiring tight regulation to maintain homeostasis and preclude disease. Using yeast and mammalian cells, we report a conserved mechanism of autophagy regulation by RNA helicase RCK family members in association with the decapping enzyme Dcp2. Under nutrient-replete conditions, Dcp2 undergoes TOR-dependent phosphorylation and associates with RCK members to form a complex with autophagy-related (ATG) mRNA transcripts, leading to decapping, degradation and autophagy suppression. Simultaneous with the induction of ATG mRNA synthesis, starvation reverses the process, facilitating ATG mRNA accumulation and autophagy induction. This conserved post-transcriptional mechanism modulates fungal virulence and the mammalian inflammasome, the latter providing mechanistic insight into autoimmunity reported in a patient with a PIK3CD/p110δ gain-of-function mutation. We propose a dynamic model wherein RCK family members, in conjunction with Dcp2, function in controlling ATG mRNA stability to govern autophagy, which in turn modulates vital cellular processes affecting inflammation and microbial pathogenesis.

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind G␣ i free of G␤␥ providing an unusual scaffold for the "G-switch" and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3⅐ G␣ i signaling module by a cell surface, seven-transmembrane receptor. AGS3 and G␣ i1 tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of ␣ 2 -adrenergic receptors or -opioid receptors reduced AGS3-RLuc⅐G␣ i1 -YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by G␤␥ sequestration with the carboxyl terminus of GRK2. G␣ i -dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to G␣ i and/or G␣ o indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both G␣␤␥ and GPR-G␣ i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

Macrophages are on the front line of host defense. They possess an array of germline-encoded pattern recognition receptors/sensors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and which activate downstream effectors/pathways to help mediate innate immune responses and host defense. Innate immune responses include the rapid induction of transcriptional networks that trigger the production of cytokines, chemokines, and cytotoxic molecules; the mobilization of cells including neutrophils and other leukocytes; the engulfment of pathogens by phagocytosis and their delivery to lysosome for degradation; and the induction of autophagy. Autophagy is a catabolic process that normally maintains cellular homeostasis in a lysosome-dependent manner, but it also functions as a cytoprotective response that intersects with a variety of general stress-response pathways. This review focuses on the intimately linked molecular mechanisms that help govern the autophagic pathway and macrophage innate immune responses.

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gi␣-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gi␣ and mammalian Inscuteable (mInsc). Gi␣ rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.The discovery of AGS3 (GPSM1) and related accessory proteins revealed unexpected functional diversity for G-protein signaling systems (8,36). AGS3 is involved in a number of different cellular activities, including asymmetric cell division during neuronal development (30), neuronal plasticity and addiction (9, 10, 12, 38, 39), autophagy (27), membrane protein trafficking (17), cardiovascular function (7), and metabolism (7). AGS3 is a multidomain protein consisting of seven tetratricopeptide repeats (TPR) in the amino-terminal portion of the protein and four G-protein regulatory (GPR) motifs in the carboxyl region of the protein. Each of the GPR motifs binds and stabilizes the GDP-bound conformation of G␣ (Gi␣, Gt␣, and Gi/o␣), essentially behaving as a guanine nucleotide dissociation inhibitor. As such, AGS3 may be complexed with up to four G␣ and function as an alternative binding partner for G␣ independently of the classical heterotrimeric G␣␤␥. Despite the clearly demonstrated function of AGS3 and the related protein LGN (GPSM2 or AGS5) in various model organisms and a fairly solid, basic biochemical understanding of the interaction of a GPR motif with G␣, the signals that operate "upstream" and/or "downstream" of AGS3 or an AGS3-Gi/o␣ complex are not well defined.AGS3 and other GPR proteins may regulate G-protein signaling directly by influencing the interaction of G␣ with G␤␥ or another G␣ binding partner. In addition, a portion of G␣ in the cell is complexed with GPR proteins to various degrees, and this interaction is regulated. Ric-8A interacts with an AGS3-Gi␣ complex in a manner somewhat analogous to the interaction of a G-protein-coupled receptor with heterotrimeric G␣␤␥, promoting nucleotide exchange and the apparent dissociation of AGS3 and Gi␣-GDP (37). The specific impact of AGS3 and other GPR proteins on signaling events is likely dependent upon where the individual protein is positioned within the c...

Background:The AGS3⅐G␣ i complex is regulated by a GPCR, but downstream signaling events are unknown. Results: Upon receptor activation, AGS3 translocates to the Golgi apparatus, where it regulates events at the TGN. Conclusion: The AGS3⅐G␣ i complex serves as a signal transducer for GPCRs. Significance: The regulated translocation of AGS3 offers unexpected mechanisms for modulating protein secretion and/or endosome recycling events at the TGN.

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to, or directly target lysosomes, the major intracellular degradative organelle. Here, we demonstrate that AGS3 (Activator of G-protein Signaling 3), a lipopolysaccharide inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GPR/GoLoco) motif stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin (mTOR) pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB (TFEB), a known activator of lysosomal gene transcription. In contrast, AGS3 deficient macrophages had increased mTOR activity, reduced TFEB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus while AGS3 deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that G␣ i nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, G␣ i proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca 2؉ that was partially sensitive to G␣ i nucleotide exchange inhibition and expression of GTP-bound G␣ i recruited Elmo1 to the plasma membrane. Reducing GDP-G␣ i nucleotide exchange, decreasing G␣ i expression, pharmacologically interrupting G␤␥ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that G␣ i remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan.