Metabolite-Sensing G Protein-Coupled Receptors-Facilitators of Diet-Related Immune Regulation.

Nutrition and the gut microbiome regulate many systems, including the immune, metabolic, and nervous systems. We propose that the host responds to deficiency (or sufficiency) of dietary and bacterial metabolites in a dynamic way, to optimize responses and survival. A family of G protein-coupled receptors (GPCRs) termed the metabolite-sensing GPCRs bind to various metabolites and transmit signals that are important for proper immune and metabolic functions. Members of this family include GPR43, GPR41, GPR109A, GPR120, GPR40, GPR84, GPR35, and GPR91. In addition, bile acid receptors such as GPR131 (TGR5) and proton-sensing receptors such as GPR65 show similar features. A consistent feature of this family of GPCRs is that they provide anti-inflammatory signals; many also regulate metabolism and gut homeostasis. These receptors represent one of the main mechanisms whereby the gut microbiome affects vertebrate physiology, and they also provide a link between the immune and metabolic systems. Insufficient signaling through one or more of these metabolite-sensing GPCRs likely contributes to human diseases such as asthma, food allergies, type 1 and type 2 diabetes, hepatic steatosis, cardiovascular disease, and inflammatory bowel diseases.

Engineering and In Vivo Applications of Riboswitches.

Riboswitches are common gene regulatory units mostly found in bacteria that are capable of altering gene expression in response to a small molecule. These structured RNA elements consist of two modular subunits: an aptamer domain that binds with high specificity and affinity to a target ligand and an expression platform that transduces ligand binding to a gene expression output. Significant progress has been made in engineering novel aptamer domains for new small molecule inducers of gene expression. Modified expression platforms have also been optimized to function when fused with both natural and synthetic aptamer domains. As this field expands, the use of these privileged scaffolds has permitted the development of tools such as RNA-based fluorescent biosensors. In this review, we summarize the methods that have been developed to engineer new riboswitches and highlight applications of natural and synthetic riboswitches in enzyme and strain engineering, in controlling gene expression and cellular physiology, and in real-time imaging of cellular metabolites and signals.

Malic enzyme 2 maintains metabolic state and anti-tumor immunity of CD8+ T cells.

The functional integrity of CD8+ T cells is closely linked to metabolic reprogramming; therefore, understanding the metabolic basis of CD8+ T cell activation and antitumor immunity could provide insights into tumor immunotherapy. Here, we report that ME2 is critical for mouse CD8+ T cell activation and immune response against malignancy. ME2 deficiency suppresses CD8+ T cell activation and anti-tumor immune response in vitro and in vivo. Mechanistically, ME2 depletion blocks the TCA cycle flux, leading to the accumulation of fumarate. Fumarate directly binds to DAPK1 and inhibits its activity by competing with ATP for binding. Notably, pharmacological inhibition of DAPK1 abolishes the anti-tumor function conferred by ME2 to CD8+ T cells. Collectively, these findings demonstrate a role for ME2 in the regulation of CD8+ T cell metabolism and effector functions as well as an unexpected function for fumarate as a metabolic signal in the inhibition of DAPK1.

L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity.

Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.

Citrulline depletion by ASS1 is required for proinflammatory macrophage activation and immune responses.

Citrulline can be converted into argininosuccinate by argininosuccinate synthetase (ASS1) in the urea cycle and the citrulline-nitric oxide cycle. However, the regulation and biological function of citrulline metabolism remain obscure in the immune system. Unexpectedly, we found that macrophage citrulline declines rapidly after interferon gamma (IFN-γ) and/or lipopolysaccharide (LPS) stimulation, which is required for efficient proinflammatory signaling activation. Mechanistically, IFN-γ and/or LPS stimulation promotes signal transducers and activators of transcription 1 (STAT1)-mediated ASS1 transcription and Janus kinase2 (JAK2)-mediated phosphorylation of ASS1 at tyrosine 87, thereby leading to citrulline depletion. Reciprocally, increased citrulline directly binds to JAK2 and inhibits JAK2-STAT1 signaling. Blockage of ASS1-mediated citrulline depletion suppresses the host defense against bacterial infection in vivo. We therefore define a central role for ASS1 in controlling inflammatory macrophage activation and antibacterial defense through depletion of cellular citrulline and, further, identify citrulline as an innate immune-signaling metabolite that engages a metabolic checkpoint for proinflammatory responses.

Metabolite-Sensing Receptor Ffar2 Regulates Colonic Group 3 Innate Lymphoid Cells and Gut Immunity.

Group 3 innate lymphoid cells (ILC3s) sense environmental signals that are critical for gut homeostasis and host defense. However, the metabolite-sensing G-protein-coupled receptors that regulate colonic ILC3s remain poorly understood. We found that colonic ILC3s expressed Ffar2, a microbial metabolite-sensing receptor, and that Ffar2 agonism promoted ILC3 expansion and function. Deficiency of Ffar2 in ILC3s decreased their in situ proliferation and ILC3-derived interleukin-22 (IL-22) production. This led to impaired gut epithelial function characterized by altered mucus-associated proteins and antimicrobial peptides and increased susceptibility to colonic injury and bacterial infection. Ffar2 increased IL-22+ CCR6+ ILC3s and influenced ILC3 abundance in colonic lymphoid tissues. Ffar2 agonism differentially activated AKT or ERK signaling and increased ILC3-derived IL-22 via an AKT and STAT3 axis. Our findings suggest that Ffar2 regulates colonic ILC3 proliferation and function, and they identify an ILC3-receptor signaling pathway modulating gut homeostasis and pathogen defense.

Dietary Fiber and Microbiota Metabolite Receptors Enhance Cognition and Alleviate Disease in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder with poorly understood etiology. AD has several similarities with other "Western lifestyle" inflammatory diseases, where the gut microbiome and immune pathways have been associated. Previously, we and others have noted the involvement of metabolite-sensing GPCRs and their ligands, short-chain fatty acids (SCFAs), in protection of numerous Western diseases in mouse models, such as Type I diabetes and hypertension. Depletion of GPR43, GPR41, or GPR109A accelerates disease, whereas high SCFA yielding diets protect in mouse models. Here, we extended the concept that metabolite-sensing receptors and SCFAs may be a more common protective mechanism against Western diseases by studying their role in AD pathogenesis in the 5xFAD mouse model. Both male and female mice were included. Depletion of GPR41 and GPR43 accelerated cognitive decline and impaired adult hippocampal neurogenesis in 5xFAD and WT mice. Lack of fiber/SCFAs accelerated a memory deficit, whereas diets supplemented with high acetate and butyrate (HAMSAB) delayed cognitive decline in 5xFAD mice. Fiber intake impacted on microglial morphology in WT mice and microglial clustering phenotype in 5xFAD mice. Lack of fiber impaired adult hippocampal neurogenesis in both W and AD mice. Finally, maternal dietary fiber intake significantly affects offspring's cognitive functions in 5xFAD mice and microglial transcriptome in both WT and 5xFAD mice, suggesting that SCFAs may exert their effect during pregnancy and lactation. Together, metabolite-sensing GPCRs and SCFAs are essential for protection against AD, and reveal a new strategy for disease prevention.Significance Statement Alzheimer's disease (AD) is one of the most common neurodegenerative diseases; currently, there is no cure for AD. In our study, short-chain fatty acids and metabolite receptors play an important role in cognitive function and pathology in AD mouse model as well as in WT mice. SCFAs also impact on microglia transcriptome, and immune cell recruitment. Out study indicates the potential of specialized diets (supplemented with high acetate and butyrate) releasing high amounts of SCFAs to protect against disease.

The crisscross between p53 and metabolism in cancer.

As the guardian of the genome, p53 is well known for its tumor suppressor function in humans, controlling cell proliferation, senescence, DNA repair and cell death in cancer through transcriptional and non-transcriptional activities. p53 is the most frequently mutated gene in human cancer, but how its mutation or depletion leads to tumorigenesis still remains poorly understood. Recently, there has been increasing evidence that p53 plays a vital role in regulating cellular metabolism as well as in metabolic adaptation to nutrient starvation. In contrast, mutant p53 proteins, especially those harboring missense mutations, have completely different functions compared to wild-type p53. In this review, we briefly summarize what is known about p53 mediating anabolic and catabolic metabolism in cancer, and in particular discuss recent findings describing how metabolites regulate p53 functions. To illustrate the variability and complexity of p53 function in metabolism, we will also review the differential regulation of metabolism by wild-type and mutant p53.

Expression and activation of the ketone body receptor HCAR2/GPR109A promotes preservation of retinal endothelial cell barrier function.

Preservation of retinal barrier function is critical to maintenance of retinal health. Therefore, it is not surprising that loss of barrier integrity is a pathologic feature common to degenerative retinal diseases such as diabetic retinopathy. Our prior studies demonstrate the importance of hydroxycarboxylic acid receptor 2/GPR109A (HCAR2/GPR109A) expression in the retinal pigment epithelium (RPE) to outer retinal barrier integrity. However, whether HCAR2/GPR109A is expressed in retinal endothelial cells and has a similar relationship to inner blood retinal barrier regulation is not known. In the current study, we examined relevance of receptor expression to endothelial cell dependent-blood retinal barrier integrity. siRNA technology was used to modulate HCAR2/GPR109A expression in human retinal endothelial cells (HRECs). Cells were cultured in the presence or absence of VEGF, a pro-inflammatory stimulus, and/or various concentrations of the HCAR2/GPR109A-specific agonist beta-hydyroxybutyrate (BHB). HCAR2/GPR109A expression was monitored by qPCR and electrical cell impedance sensing (ECIS) was used to evaluate barrier function. Complementary in vivo studies were conducted in wildtype and HCAR2/GPR109A knockout mice treated intraperitoneally with lipopolysaccharide and/or BHB. Vascular leakage was monitored using fluorescein angiography and Western blot analyses of albumin extravasation. Additionally, retinal function was evaluated by OptoMotry. Decreased (siRNA knockdown) or absent (gene knockout) HCAR2/GPR109A expression was associated with impaired barrier function both in vitro and in vivo. BHB treatment provided some protection, limiting disruptions in retinal barrier integrity and function; an effect that was found to be receptor (HCAR2/GPR109A)-dependent. Collectively, the present studies support a key role for HCAR2/GPR109A in regulating blood-retinal barrier integrity and highlight the therapeutic potential of the receptor toward preventing and treating retinal diseases such as diabetic retinopathy in which compromised barrier function is paramount.

Simultaneous probing of dual intracellular metabolites (ATP and paramylon) in live microalgae using graphene oxide/aptamer nanocomplex.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3 nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10 mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells.

Metabolite sensing and signaling in cancer.

Metabolites are not only substrates in metabolic reactions, but also signaling molecules controlling a wide range of cellular processes. Discovery of the oncometabolite 2-hydroxyglutarate provides an important link between metabolic dysfunction and cancer, unveiling the signaling function of metabolites in regulating epigenetic and epitranscriptomic modifications, genome integrity, and signal transduction. It is now known that cancer cells remodel their metabolic network to support biogenesis, caused by or resulting in the dysregulation of various metabolites. Cancer cells can sense alterations in metabolic intermediates to better coordinate multiple biological processes and enhance cell metabolism. Recent studies have demonstrated that metabolite signaling is involved in the regulation of malignant transformation, cell proliferation, epithelial-to-mesenchymal transition, differentiation blockade, and cancer stemness. Additionally, intercellular metabolite signaling modulates inflammatory response and immunosurveillance in the tumor microenvironment. Here, we review recent advances in cancer-associated metabolite signaling. An in depth understanding of metabolite signaling will provide new opportunities for the development of therapeutic interventions that target cancer.

GPR43 regulates sodium butyrate-induced angiogenesis and matrix remodeling.

Butyrate is a short-chain fatty acid (SCFA) derived from microbiota and is involved in a range of cell processes in a concentration-dependent manner. Low concentrations of sodium butyrate (NaBu) were shown to be proangiogenic. However, the mechanisms associated with these effects are not yet fully known. Here, we investigated the contribution of the SCFA receptor GPR43 in the proangiogenic effects of local treatment with NaBu and its effects on matrix remodeling using the sponge-induced fibrovascular tissue model in mice lacking the Gpr43 gene (Gpr43-KO) and the wild-type (WT) mice. We demonstrated that NaBu (0.2 mM intraimplant) treatment enhanced the neovascularization process, blood flow, and VEGF levels in a GPR43-dependent manner in the implants. Moreover, NaBu was able to modulate matrix remodeling aspects of the granulation tissue such as proteoglycan production, collagen deposition, and α-smooth muscle actin (α-SMA) expression in vivo, besides increasing transforming growth factor (TGF)-ß1 levels in the fibrovascular tissue, in a GPR43-dependent manner. Interestingly, NaBu directly stimulated L929 murine fibroblast migration and TGF-ß1 and collagen production in vitro. GPR43 was found to be expressed in human dermal fibroblasts, myofibroblasts, and endothelial cells. Overall, our findings evidence that the metabolite-sensing receptor GPR43 contributes to the effects of low dose of NaBu in inducing angiogenesis and matrix remodeling during granulation tissue formation. These data provide important insights for the proposition of new therapeutic approaches based on NaBu, beyond the highly explored intestinal, anti-inflammatory, and anticancer purposes, as a local treatment to improve tissue repair, particularly, by modulating granulation tissue components.NEW & NOTEWORTHY Our data show the contribution of the metabolite-sensing receptor GPR43 in the effects of low dose of sodium butyrate (NaBu) on stimulating angiogenesis and extracellular matrix remodeling in a model of granulation tissue formation in mice. We also show that human dermal fibroblasts, myofibroblasts, and endothelial cells express the receptor GPR43. These data provide important insights for the use of NaBu in local therapeutic approaches applicable to tissue repair in sites other than the intestine.

A highly sensitive molecular structural probe applied to in situ biosensing of metabolites using PEDOT:PSS.

A large amount of research within organic biosensors is dominated by organic electrochemical transistors (OECTs) that use conducting polymers such as poly(3,4-ethylene dioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS). Despite the recent advances in OECT-based biosensors, the sensing is solely reliant on the amperometric detection of the bioanalytes. This is typically accompanied by large undesirable parasitic electrical signals from the electroactive components in the electrolyte. Herein, we present the use of in situ resonance Raman spectroscopy to probe subtle molecular structural changes of PEDOT:PSS associated with its doping level. We demonstrate how such doping level changes of PEDOT:PSS can be used, for the first time, on operational OECTs for sensitive and selective metabolite sensing while simultaneously performing amperometric detection of the analyte. We test the sensitivity by molecularly sensing a lowest glucose concentration of 0.02 mM in phosphate-buffered saline solution. By changing the electrolyte to cell culture media, the selectivity of in situ resonance Raman spectroscopy is emphasized as it remains unaffected by other electroactive components in the electrolyte. The application of this molecular structural probe highlights the importance of developing biosensing probes that benefit from high sensitivity of the material's structural and electrical properties while being complimentary with the electronic methods of detection.

Role of a hairpin-stabilized pause in the Escherichia coli thiC riboswitch function.

Transcriptional pauses have been reported in bacterial riboswitches and, in some cases, their specific positioning has been shown to be important for gene regulation. Here, we show that a hairpin structure in the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch is involved in transcriptional pausing and ligand sensitivity. Using in vitro transcription kinetic experiments, we show that all three major transcriptional pauses in the thiC riboswitch are affected by NusA, a transcriptional factor known to stimulate hairpin-stabilized pauses. Using a truncated region of the riboswitch, we isolated the hairpin structure responsible for stabilization of the most upstream pause. Destabilization of this structure led to a weaker pause and a decreased NusA effect. In the context of the full-length riboswitch, this same mutation also led to a weaker pause, as well as a decreased TPP binding affinity. Our work suggests that RNA structures involved in transcriptional pausing in riboswitches are important for ligand sensitivity, most likely by increasing the time allowed to the ligand for binding to the riboswitch.

Recent Progress in Lab-On-a-Chip Systems for the Monitoring of Metabolites for Mammalian and Microbial Cell Research.

Lab-on-a-chip sensing technologies have changed how cell biology research is conducted. This review summarises the progress in the lab-on-a-chip devices implemented for the detection of cellular metabolites. The review is divided into two subsections according to the methods used for the metabolite detection. Each section includes a table which summarises the relevant literature and also elaborates the advantages of, and the challenges faced with that particular method. The review continues with a section discussing the achievements attained due to using lab-on-a-chip devices within the specific context. Finally, a concluding section summarises what is to be resolved and discusses the future perspectives.

In vivo biosensors: mechanisms, development, and applications.

In vivo biosensors can recognize and respond to specific cellular stimuli. In recent years, biosensors have been increasingly used in metabolic engineering and synthetic biology, because they can be implemented in synthetic circuits to control the expression of reporter genes in response to specific cellular stimuli, such as a certain metabolite or a change in pH. There are many types of natural sensing devices, which can be generally divided into two main categories: protein-based and nucleic acid-based. Both can be obtained either by directly mining from natural genetic components or by engineering the existing genetic components for novel specificity or improved characteristics. A wide range of new technologies have enabled rapid engineering and discovery of new biosensors, which are paving the way for a new era of biotechnological progress. Here, we review recent advances in the design, optimization, and applications of in vivo biosensors in the field of metabolic engineering and synthetic biology.

Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae.

Biophysical Approaches to Bacterial Gene Regulation by Riboswitches.

The last decade has witnessed the discovery of a variety of non-coding RNA sequences that perform a broad range of crucial biological functions. Among these, the ability of certain RNA sequences, so-called riboswitches, has attracted considerable interest. Riboswitches control gene expression in response to the concentration of particular metabolites to which they bind without the need for any protein. These RNA switches not only need to adopt a very specific tridimensional structure to perform their function, but also their sequence has been evolutionary optimized to recognize a particular metabolite with high affinity and selectivity. Thus, riboswitches offer a unique opportunity to get fundamental insights into RNA plasticity and how folding dynamics and ligand recognition mechanisms have been efficiently merged to control gene regulation. Because riboswitch sequences have been mostly found in bacterial organisms controlling the expression of genes associated to the synthesis, degradation or transport of crucial metabolites for bacterial survival, they offer exciting new routes for antibiotic development in an era where bacterial resistance is more than ever challenging conventional drug discovery strategies. Here, we give an overview of the architecture, diversity and regulatory mechanisms employed by riboswitches with particular emphasis on the biophysical methods currently available to characterise their structure and functional dynamics.

Tuning a riboswitch response through structural extension of a pseudoknot.

Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ1 class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem-loop structure within its 3'-terminal region immediately upstream of the Shine-Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ1 riboswitch folding from multiple structural perspectives, we reveal that the extra stem-loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem-loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.

Role of lysine binding residues in the global folding of the lysC riboswitch.

Riboswitches regulate gene expression by rearranging their structure upon metabolite binding. The lysine-sensing lysC riboswitch is a rare example of an RNA aptamer organized around a 5-way helical junction in which ligand binding is performed exclusively through nucleotides located at the junction core. We have probed whether the nucleotides involved in ligand binding play any role in the global folding of the riboswitch. As predicted, our findings indicate that ligand-binding residues are critical for the lysine-dependent gene regulation mechanism. We also find that these residues are not important for the establishment of key magnesium-dependent tertiary interactions, suggesting that folding and ligand recognition are uncoupled in this riboswitch for the formation of specific interactions. However, FRET assays show that lysine binding results in an additional conformational change, indicating that lysine binding may also participate in a specific folding transition. Thus, in contrast to helical junctions being primary determinants in ribozymes and rRNA folding, we speculate that the helical junction of the lysine-sensing lysC riboswitch is not employed as structural a scaffold to direct global folding, but rather has a different role in establishing RNA-ligand interactions required for riboswitch regulation. Our work suggests that helical junctions may adopt different functions such as the coordination of global architecture or the formation of specific ligand binding site.