Biochemistry, Cell Biology, and Pathophysiology of the Innate Immune cGAS-cGAMP-STING Pathway.

In the decade since the discovery of the innate immune cyclic GMP-AMP synthase (cGAS)-2'3'-cyclic GMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway, its proper activation and dysregulation have been rapidly implicated in many aspects of human disease. Understanding the biochemical, cellular, and regulatory mechanisms of this pathway is critical to developing therapeutic strategies that either harness it to boost defense or inhibit it to prevent unwanted inflammation. In this review, we first discuss how the second messenger cGAMP is synthesized by cGAS in response to double-stranded DNA and cGAMP's subsequent activation of cell-type-dependent STING signaling cascades with differential physiological consequences. We then review how cGAMP as an immunotransmitter mediates tightly controlled cell-cell communication by being exported from producing cells and imported into responding cells via cell-type-specific transporters. Finally, we review mechanisms by which thecGAS-cGAMP-STING pathway responds to different sources of mislocalized double-stranded DNA in pathogen defense, cancer, and autoimmune diseases.

PELI2 is a negative regulator of STING signaling that is dynamically repressed during viral infection.

The innate immune cGAS-STING pathway is activated by cytosolic double-stranded DNA (dsDNA), a ubiquitous danger signal, to produce interferon, a potent anti-viral and anti-cancer cytokine. However, STING activation must be tightly controlled because aberrant interferon production leads to debilitating interferonopathies. Here, we discover PELI2 as a crucial negative regulator of STING. Mechanistically, PELI2 inhibits the transcription factor IRF3 by binding to phosphorylated Thr354 and Thr356 on the C-terminal tail of STING, leading to ubiquitination and inhibition of the kinase TBK1. PELI2 sets a threshold for STING activation that tolerates low levels of cytosolic dsDNA, such as that caused by silenced TREX1, RNASEH2B, BRCA1, or SETX. When this threshold is reached, such as during viral infection, STING-induced interferon production temporarily downregulates PELI2, creating a positive feedback loop allowing a robust immune response. Lupus patients have insufficient PELI2 levels and high basal interferon production, suggesting that PELI2 dysregulation may drive the onset of lupus and other interferonopathies.

IFN-Independent STING Signaling: Friend or Foe?

In this issue, Wu et al. demonstrate the importance of the neglected interferon (IFN)-independent STING signaling axis in mice. They find that although this axis is important for antiviral HSV-1 resistance, it has a pro-cancer role by promoting T cell death.

LRRC8A:C/E Heteromeric Channels Are Ubiquitous Transporters of cGAMP.

Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter exported by diseased cells and imported into host cells to activate the innate immune STING pathway. We previously identified SLC19A1 as a cGAMP importer, but its use across human cell lines is limited. Here, we identify LRRC8A heteromeric channels, better known as volume-regulated anion channels (VRAC), as widely expressed cGAMP transporters. LRRC8A forms complexes with LRRC8C and/or LRRC8E, depending on their expression levels, to transport cGAMP and other 2'3'-cyclic dinucleotides. In contrast, LRRC8D inhibits cGAMP transport. We demonstrate that cGAMP is effluxed or influxed via LRRC8 channels, as dictated by the cGAMP electrochemical gradient. Activation of LRRC8A channels, which can occur under diverse stresses, strongly potentiates cGAMP transport. We identify activator sphingosine 1-phosphate and inhibitor DCPIB as chemical tools to manipulate channel-mediated cGAMP transport. Finally, LRRC8A channels are key cGAMP transporters in resting primary human vasculature cells and universal human cGAMP transporters when activated.

SLC19A1 Is an Importer of the Immunotransmitter cGAMP.

2'3'-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral stimulator of interferon genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen, we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMP's role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein Binding Activities.

UNLABELLED: The HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation. IMPORTANCE: The HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the mechanisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the mutant proteins showed better binding to Env CTs than the WT proteins, and CT binding correlated with MA trimerization. Our results suggest that multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.

Analysis of HIV-1 Gag protein interactions via biotin ligase tagging.

UNLABELLED: We have examined the interactions of wild-type (WT) and matrix protein-deleted (ΔMA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase-tagging approach. To do so, WT and ΔMA PrGag proteins were tagged with the Escherichia coli promiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves as well as WT PrGag proteins in trans. Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, ΔMA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting that the presence of MA made Env inaccessible to biotinylation. We also identified over 50 cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA-processing proteins. The identification of these biotinylated proteins offers new insights into HIV-1 Gag protein trafficking and activities and provides new potential targets for antiviral interference. IMPORTANCE: We have employed a novel strategy to analyze the interactions of the HIV-1 structural Gag proteins, which involved tagging wild-type and mutant Gag proteins with a biotin ligase. Expression of the tagged proteins in cells allowed us to analyze proteins that came in close proximity to the Gag proteins as they were synthesized, transported, assembled, and released from cells. The tagged proteins biotinylated proteins encoded by the HIV-1 pol gene and neighbor Gag proteins, but, surprisingly, only the mutant Gag protein biotinylated the HIV-1 Envelope protein. We also identified over 50 cellular proteins that were biotinylated, including membrane and cytoskeletal proteins and proteins involved in lipid metabolism, nuclear import, translation, and RNA processing. Our results offer new insights into HIV-1 Gag protein trafficking and activities and provide new potential targets for antiviral interference.

Analysis of quinolinequinone reactivity, cytotoxicity, and anti-HIV-1 properties.

We have analyzed a set of quinolinequinones with respect to their reactivities, cytotoxicities, and anti-HIV-1 properties. Most of the quinolinequinones were reactive with glutathione, and several acted as sulfhydryl crosslinking agents. Quinolinequinones inhibited binding of the HIV-1 matrix protein to RNA to varying degrees, and several quinolinequinones showed the capacity to crosslink HIV-1 matrix proteins in vitro, and HIV-1 structural proteins in virus particles. Cytotoxicity assays yielded quinolinequinone CC50 values in the low micromolar range, reducing the potential therapeutic value of these compounds. However, one compound, 6,7-dichloro-5,8-quinolinequinone potently inactivated HIV-1, suggesting that quinolinequinones may prove useful in the preparation of inactivated virus vaccines or for other virucidal purposes.

Investigation into the Sensory Properties of Plant-Based Eggs, as Well as Acceptance, Emotional Response, and Use.

Consumers have become interested in plant-based alternatives to animal-based products. One of the under-studied alternatives is plant-based eggs (PBEs). This research investigated PBEs relative to conventional eggs and tofu scramble-another plant-based alternative. Firstly, participants (n = 93) completed a word association task asking them about PBEs. Participants then evaluated the different food samples using hedonic scales, check-all-that-apply (CATA), and temporal check-all-that-apply (TCATA), as well as identified their emotional response and proposed use for PBEs. Participants were interested in plant-based alternatives, including PBEs, but they were concerned about the sensory properties. When they evaluated the different samples, the flavour and texture of the PBEs were disliked in comparison to the eggs. This result may be due to the beany, bitterness, and off-flavour attributes associated with the PBEs. Participants also associated the PBEs with negative emotions. The liking of tofu scramble was not significantly different from the eggs, and the eggs and tofu scramble were mainly associated with positive emotions. During the TCATA evaluation, the participants focused on the flavour attributes of PBEs, while their evaluation of the eggs was dominated by the textural attributes. Whether following a plant-based diet or not, consumers are interested in PBEs, but the sensory properties of PBEs need to be improved before they are willing to adopt them into their diet. This study is one of the first to evaluate the sensory properties of PBEs, as well as consumers' emotional response to them and their attitudes about PBEs.

A Prospective Review of the Sensory Properties of Plant-Based Dairy and Meat Alternatives with a Focus on Texture.

Consumers are interested in plant-based alternatives (PBAs) to dairy and meat products, and as such, the food industry is responding by developing a variety of different plant-based food items. For these products to be successful, their textural properties must be acceptable to consumers. These textural properties need to be thoroughly investigated using different sensory methodologies to ensure consumer satisfaction. This review paper aims to summarize the various textural properties of PBAs, as well as to discuss the sensory methodologies that can be used in future studies of PBAs. PBAs to meat have been formulated using a variety of production technologies, but these products still have textural properties that differ from animal-based products. Most dairy and meat alternatives attempt to mimic their conventional counterparts, yet sensory trials rarely compare the PBAs to their meat or dairy counterparts. While most studies rely on consumers to investigate the acceptability of their products' textural properties, future studies should include dynamic sensory methodologies, and attribute diagnostics questions to help product developers characterize the key sensory properties of their products. Studies should also indicate whether the product is meant to mimic a conventional product and should define the target consumer segment (ex. flexitarian, vegan) for the product. The importance of textural properties to PBAs is repeatedly mentioned in the literature and thus should be thoroughly investigated using robust sensory methodologies.

Perceptions of plant-based fish among Atlantic Canadians.

There is an increasing demand for plant-based alternatives by individuals living in the Western world. One of the newer plant-based alternatives is plant-based fish and seafood (PBFs). This study aimed to investigate individuals' opinions and attitudes toward PBFs, as well as evaluate the effect of involvement in the fishing industry on the participants' attitudes. Participants (n = 183) were asked to answer questions about their perceptions of PBFs. Participants believed that PBFs were environment-friendly and were interested in trying PBFs but were concerned about the taste and texture of PBFs. Although participants were likely to try PBFs, they were less likely to add them to their regular diet. After reading messages about the benefits of PBFs in this study, participants' willingness to try PBFs and add PBFs into their regular diet increased. In addition, those who worked in the fishing industry or had high food neophobic scores did not believe that PBFs would taste like conventional fish and seafood products. Future studies should investigate the attitudes of individuals living in different regions and investigate whether exposure to PBFs affects consumer perceptions of the food product. PRACTICAL APPLICATION: Consumer demand for new plant-based products is increasing, but before new products can be released, participants' attitudes and perceptions need to be evaluated. Plant-based alternatives to fish and seafood are a new food product, and therefore, participants' attitudes toward them need to be investigated. It was found that the individuals were more willing to try plant-based fish and seafood. In addition, they were more likely to incorporate them into their diet after reading about the nutritional benefits and sustainability of PBFs.

Evaluation of the sensory properties of thickened and protein-enhanced ice cream using check-all-that-apply and temporal check-all-that-apply.

Ice cream formulations with varying amounts of added whey protein were created for those living with dysphagia in long-term care facilities (LTCs) to improve protein and fluid intake. The samples of thickened ice cream included a control (0% whey protein [WP]) and formulations with 6% (6WP), 8% (8WP), 10% (10WP), 12% (12WP) and 14% (14WP) added whey protein by volume. The consistency of the samples was assessed using the International Dysphagia Diet Standardization Initiative (IDDSI) Spoon Tilt Test, a sensory trial (n = 102) using hedonic scales and check-all-that-apply (CATA) and another sensory trial (n = 96) using temporal check-all-that-apply (TCATA). The whey protein increased the acceptability of the thickened ice cream except for the 12WP and 14WP formulations. The formulations with higher amounts of whey protein were associated with bitterness, custard/eggy flavor, and mouthcoating. The TCATA identified that the addition of whey protein led to slippery, gritty, and grainy attributes being perceived in the thickened ice cream. The study identified that 10% whey protein by volume can be added to thickened ice cream without impacting its' acceptability and the 6WP, 8WP, and 10WP formulations were liked significantly more than the control (without whey protein).

Effect of Piperine on Saltiness Perception.

Chemical irritants, like piperine, have the potential to increase human perception of tastes and odours, including saltiness. This cross-modal interaction could help the food industry develop new salt-reduced food products that maintain their salty taste. The objective of this study was: firstly, to determine the detection threshold of piperine (n = 72), secondly to evaluate piperine's influence on saltiness perception in model solutions (n = 78), and lastly to identify piperine's effect on sensory perception of low sodium soup using temporal check-all-that-apply (TCATA; n = 75). The group mean of the individual threshold was 0.55 ± 0.15 ppm. Piperine increased the saltiness perception of the model solutions, but it also increased the bitterness and decreased the sweetness of the solutions. The piperine significantly increased the saltiness intensity of the soups (evaluated using a generalized labelled magnitude), but during the TCATA task, the salty attribute was selected less for the soup with piperine than the control (based on the average proportion of selection). The TCATA indicated that the peppery attribute dominated the participants' perception of the soup with piperine. More studies are needed to assess piperine's cross-modal interactions.

Human SLC46A2 Is the Dominant cGAMP Importer in Extracellular cGAMP-Sensing Macrophages and Monocytes.

Administration of exogenous CDNs to activate the cGAMP-STING pathway is a promising therapeutic strategy to unleash the full potential of cancer immunotherapy. This strategy mirrors the role of endogenous extracellular cGAMP, an immunotransmitter that is transferred from cancer cells to cGAMP-sensing cells in the host, promoting immunity. However, the CDN import mechanisms used by host cells within tumors remain unknown. Here we identified the protein SLC46A2 as the dominant cGAMP importer in primary human monocytes. Furthermore, we discovered that monocytes and M1-polarized macrophages directly sense tumor-derived extracellular cGAMP in murine tumors. Finally, we demonstrated that SLC46A2 is the dominant cGAMP importer in monocyte-derived macrophages. Together, we provide the first cellular and molecular mechanisms of cGAMP as an immunotransmitter, paving the way for effective STING pathway therapeutics.

Analysis of K-Ras Interactions by Biotin Ligase Tagging.

BACKGROUND: Mutations of the human K-Ras 4B (K-Ras) G protein are associated with a significant proportion of all human cancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network. MATERIALS AND METHODS: We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximity-dependent fashion, and proteins are identified via mass spectrometry (MS) sequencing. RESULTS: In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers. CONCLUSION: Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks.