Signaling Platforms Research Articles

Endosomes serve as signaling platforms for RIG-I ubiquitination and activation.

RIG-I-like receptors (RLRs) are cytosolic RNA sensors critical for antiviral immunity. RLR activation is regulated by polyubiquitination and oligomerization following RNA binding. Yet, little is known about how RLRs exploit subcellular organelles to facilitate their posttranslational modifications and activation. Endosomal adaptor TAPE regulates the endosomal TLR and cytosolic RLR pathways. The potential interplay between RIG-I signaling and endosomes has been explored. Here, we report that endosomes act as platforms for facilitating RIG-I polyubiquitination and complex formation. RIG-I was translocated onto endosomes to form signaling complexes upon activation. Ablation of endosomes impaired RIG-I signaling to type I IFN activation. TAPE mediates the interaction and polyubiquitination of RIG-I and TRIM25. TAPE-deficient myeloid cells were defective in type I IFN activation upon RNA ligand and virus challenges. Myeloid TAPE deficiency increased the susceptibility to RNA virus infection in vivo. Our work reveals endosomes as signaling platforms for RIG-I activation and antiviral immunity.

New mutations in the core Schizosaccharomyces pombe spindle pole body scaffold Ppc89 reveal separable functions in regulating cell division.

Centrosomes and spindle pole bodies (SPB) are important for mitotic spindle formation and also serve as signaling platforms. In the fission yeast Schizosaccharomyces pombe, genetic ablation and high-resolution imaging indicate that the ɑ-helical Ppc89 is central to SPB structure and function. Here, we developed and characterized conditional and truncation mutants of ppc89. Alleles with mutations in two predicted ɑ-helices near the C-terminus were specifically defective in anchoring Sid4, the scaffold for the septation initiation network (SIN), and proteins dependent on Sid4 (Cdc11, Dma1, Mto1 and Mto2). Artificial tethering of Sid4 to the SPB fully rescued these ppc89 mutants. Another ppc89 allele had mutations located throughout the coding region. While this mutant was also defective in Sid4 anchoring, it displayed additional defects including fragmented SPBs and forming and constricting a second cytokinetic ring in one daughter cell. These defects were shared with a ppc89 allele truncated of the most C-terminal predicted ɑ-helices that is still able to recruit Sid4 and the SIN. We conclude that Ppc89 not only tethers the SIN to the SPB but is also necessary for the integrity of the SPB and faithful coordination of cytokinesis with mitosis.

Lipid Compositions of Liquid-Ordered and Liquid-Disordered Phases in Ternary Membranes of Sphingomyelin, Cholesterol, and Dioleoylphosphatidylcholine Determined by 2H NMR: Stearoyl-Sphingomyelin Compared with Its Palmitoyl Counterpart.

Sphingomyelin (SM) and cholesterol are the major lipids in the signaling platforms of cell membranes, known as lipid rafts. In particular, SM with a stearoyl chain (C18-SM) is abundant in specific tissues such as the brain, the most cholesterol-rich organ, whereas the distribution of palmitoyl (C16)-SM is ubiquitous. Here, we reveal the differences between palmitoyl- and stearoyl-SM in lipid-lipid interactions based on the tie lines obtained from the 2H solid-state NMR spectra of bilayer systems composed of SM/dioleoylphosphatidylcholine/cholesterol 33:33:33 and 40:40:20. Lipid probes carrying position-selective deuterations, 10',10'-d2-SM, 24-d1-cholesterol, and 6″,6″-d2-dioleoyl-phosphatidylcholine, were incorporated into the membranes. 2H NMR peaks from these probes in the membranes directly provide the lipid compositions of the liquid-ordered (Lo) and liquid-disordered (Ld) regions. Without using bulky fluorescent groups, these probes allow us to obtain the end points of the tie lines in a ternary phase diagram based on the lever rule. Consequently, the tie lines of the stearoyl-SM membranes were steeper than those of the palmitoyl-SM membranes, indicating that cholesterol content was higher in the Lo domains of stearoyl-SM, regardless of the total concentration of unsaturated phospholipids. When comparing the content of unsaturated lipids in the Lo domain, the stearoyl-SM membranes had a higher content than palmitoyl-SM membranes. These results revealed that stearoyl-SM is suitable for stabilizing biologically functional microdomains in cholesterol-rich organs, whereas palmitoyl-SM may be better suited for stabilizing domains in tissue membranes with normal cholesterol content. The small but significant differences in the lipid interactions between stearoyl-SM and palmitoyl-SM may be related to the spatiotemporal formation of functional domains in biological environments.

The Evolution of NLR Inflammasome and Its Mediated Pyroptosis in Metazoa.

Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) inflammasomes are multiprotein signaling platforms that control the inflammatory response and coordinate antimicrobial defense. In the present study, the distribution of NLR, Caspase-1, and gasdermin (GSDM) homologues and their structural characteristics and evolutionary relationships were systematically analyzed in metazoa according to the genomes of species. In invertebrates, there were only NLRC and/or NLRD presented from sponge to amphioxus, and according to the evolutionary tree, NLR from sponge located in the most primitive position. Caspase-1 existed in some metazoan phyla (Brachiopoda, Ectoprocta, Arthropoda, Mollusca, Annelia, Nematoda, Platyelminthes, Coelenterate, and Porifera) and its activation sites were relatively conserved. The amino acid sequences and three-dimensional structures of N-terminal CARD/Death domain of NLR and Caspase-1 were similar in species from sponge to human. NLR and Caspase-1 co-existed in species of Brachiopoda, Mollusca, Annelia, Coelenterate, and Porifera. There was only GSDME or PJVK found in some phyla of invertebrates and their cleavage sites were conserved (DxxD). And it was predicted that the NLR inflammasome in inducing pyroptosis could occur in species of Brachiopoda, Mollusca, Annelia, and Coelenterate. These studies indicated that NLR inflammasome emerged early in sponges of metazoa, and NLR inflammasome in inducing pyroptosis first appeared in Coelenterate, suggesting that inflammasome and its mediated pyroptosis had existed in the early stage of metazoa, but they had been lost in many species during evolution.

Redox Signaling in Endosomes Using the Example of EGF Receptors: A Graphical Review.

Early endosomes represent first-line sorting compartments or even organelles for internalized molecules. They enable the transport of molecules or ligands to other compartments of the cell, such as lysosomes, for degradation or recycle them back to the membrane by various mechanisms. Moreover, early endosomes function as signaling and scaffolding platforms to initiate or prolong distinct signaling pathways. Accordingly, early endosomes have to be recognized as either part of a degradation or recycling pathway. The physical proximity of many ligand-binding receptors with other membrane-bound proteins or complexes such as NADPH oxidases may result in an interaction of second messengers, like reactive oxygen species (ROS) and early endosomes, that promote the correct recognition of individual early endosomes. In fact, redoxosomes comprise an endosomal subsection of signaling endosomes. One example of such potential interaction is epidermal growth factor receptor (EGFR) signaling. Here we summarize recent findings on EGFR signaling as a well-studied example for receptor trafficking and trans-activation and illustrate the interplay between cellular and endosomal ROS.

A20 intrinsically influences human effector T-cell survival and function by regulating both NF-κB and JNK signaling.

A20 is a dual-function ubiquitin-editing enzyme that maintains immune homeostasis by restraining inflammation. Although A20 serves a similar negative feedback function for T-cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T-cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-κB and c-Jun/AP-1. Utilizing WT and A20 knockout Jurkat T cells, we found that A20 is required to negatively regulate NF-κB and JNK. Utilizing a novel set of A20 mutants in NF-κB and AP-1-driven reporter systems, we discovered the ZnF7 domain is crucial for negative regulatory capacity, while deubiquitinase activity is dispensable. Successful inactivation of A20 in human primary effector T cells congruently conferred sustained NF-κB and JNK signaling, including enhanced upregulation of activation markers, and increased secretion of several cytokines including IL-9. Finally, loss of A20 in primary human T cells resulted in decreased sensitivity to restimulation-induced cell death and increased sensitivity to cytokine withdrawal-induced death. These findings demonstrate the importance of A20 in maintaining T-cell homeostasis via negative regulation of both NF-κB and JNK signaling.

LYCHOS is a human hybrid of a plant-like PIN transporter and a GPCR

Lysosomes have crucial roles in regulating eukaryotic metabolism and cell growth by acting as signalling platforms to sense and respond to changes in nutrient and energy availability1. LYCHOS (GPR155) is a lysosomal transmembrane protein that functions as a cholesterol sensor, facilitating the cholesterol-dependent activation of the master protein kinase mechanistic target of rapamycin complex 1 (mTORC1)2. However, the structural basis of LYCHOS assembly and activity remains unclear. Here we determine several high-resolution cryo-electron microscopy structures of human LYCHOS, revealing a homodimeric transmembrane assembly of a transporter-like domain fused to a G-protein-coupled receptor (GPCR) domain. The class B2-like GPCR domain is captured in the apo state and packs against the surface of the transporter-like domain, providing an unusual example of a GPCR as a domain in a larger transmembrane assembly. Cholesterol sensing is mediated by a conserved cholesterol-binding motif, positioned between the GPCR and transporter domains. We reveal that the LYCHOS transporter-like domain is an orthologue of the plant PIN-FORMED (PIN) auxin transporter family, and has greater structural similarity to plant auxin transporters than to known human transporters. Activity assays support a model in which the LYCHOS transporter and GPCR domains coordinate to sense cholesterol and regulate mTORC1 activation.

Lysosomes in the immunometabolic reprogramming of immune cells in atherosclerosis.

Lysosomes have a central role in the disposal of extracellular and intracellular cargo and also function as metabolic sensors and signalling platforms in the immunometabolic reprogramming of macrophages and other immune cells in atherosclerosis. Lysosomes can rapidly sense the presence of nutrients within immune cells, thereby switching from catabolism of extracellular material to the recycling of intracellular cargo. Such a fine-tuned degradative response supports the generation of metabolic building blocks through effectors such as mTORC1 or TFEB. By coupling nutrients to downstream signalling and metabolism, lysosomes serve as a crucial hub for cellular function in innate and adaptive immune cells. Lysosomal dysfunction is now recognized to be a hallmark of atherogenesis. Perturbations in nutrient-sensing and signalling have profound effects on the capacity of immune cells to handle cholesterol, perform phagocytosis and efferocytosis, and limit the activation of theinflammasome and other inflammatory pathways. Strategies to improve lysosomal function hold promise as novel modulators of the immunoinflammatory response associated with atherosclerosis. In this Review, we describe the crosstalk between lysosomal biology and immune cell function and polarization, with a particular focus on cellular immunometabolic reprogramming in the context of atherosclerosis.

Biochemical Pathways Delivering Distinct Glycosphingolipid Patterns in MDA-MB-231 and MCF-7 Breast Cancer Cells.

The complex structure of glycosphingolipids (GSLs) supports their important role in cell function as modulators of growth factor receptors and glutamine transporters in plasma membranes. The aberrant composition of clustered GSLs within signaling platforms, so-called lipid rafts, inevitably leads to tumorigenesis due to disturbed growth factor signal transduction and excessive uptake of glutamine and other molecules needed for increased energy and structural molecule cell supply. GSLs are also involved in plasma membrane processes such as cell adhesion, and their transition converts cells from epithelial to mesenchymal with features required for cell migration and metastasis. Glutamine activates the mechanistic target of rapamycin complex 1 (mTORC1), resulting in nucleotide synthesis and proliferation. In addition, glutamine contributes to the cancer stem cell GD2 ganglioside-positive phenotype in the triple-negative breast cancer cell line MDA-MB-231. Thieno[2,3-b]pyridine derivative possesses higher cytotoxicity against MDA-MB-231 than against MCF-7 cells and induces a shift to aerobic metabolism and a decrease in S(6)nLc4Cer GSL-positive cancer stem cells in the MDA-MB-231 cell line. In this review, we discuss findings in MDA-MB-231, MCF-7, and other breast cancer cell lines concerning their differences in growth factor receptors and recent knowledge of the main biochemical pathways delivering distinct glycosphingolipid patterns during tumorigenesis and therapy.

Inflammasome components as new therapeutic targets in inflammatory disease.

Inflammation drives pathology in many human diseases for which there are no disease-modifying drugs. Inflammasomes are signalling platforms that can induce pathological inflammation and tissue damage, having potential as an exciting new class of drug targets. Small-molecule inhibitors of the NLRP3 inflammasome that are now in clinical trials have demonstrated proof of concept that inflammasomes are druggable, and so drug development programmes are now focusing on other key inflammasome molecules. In this Review, we describe the potential of inflammasome components as candidate drug targets and the novel inflammasome inhibitors that are being developed. We discuss how the signalling biology of inflammasomes offers mechanistic insights for therapeutic targeting. We also discuss the major scientific and technical challenges associated with drugging these molecules during preclinical development and clinical trials.

Guidelines for naming and studying plasma membrane domains in plants.

Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.

Glutamate, Gangliosides, and the Synapse: Electrostatics at Work in the Brain.

The synapse is a piece of information transfer machinery replacing the electrical conduction of nerve impulses at the end of the neuron. Like many biological mechanisms, its functioning is heavily affected by time constraints. The solution selected by evolution is based on chemical communication that, in theory, cannot compete with the speed of nerve conduction. Nevertheless, biochemical and biophysical compensation mechanisms mitigate this intrinsic weakness: (i) through the high concentrations of neurotransmitters inside the synaptic vesicles; (ii) through the concentration of neurotransmitter receptors in lipid rafts, which are signaling platforms; indeed, the presence of raft lipids, such as gangliosides and cholesterol, allows a fine tuning of synaptic receptors by these lipids; (iii) through the negative electrical charges of the gangliosides, which generate an attractive (for cationic neurotransmitters, such as serotonin) or repulsive (for anionic neurotransmitters, such as glutamate) electric field. This electric field controls the flow of glutamate in the tripartite synapse involving pre- and post-synaptic neurons and the astrocyte. Changes in the expression of brain gangliosides can disrupt the functioning of the glutamatergic synapse, causing fatal diseases, such as Rett syndrome. In this review, we propose an in-depth analysis of the role of gangliosides in the glutamatergic synapse, highlighting the primordial and generally overlooked role played by the electric field of synaptic gangliosides.

Abstract Mo020: Loss of TRAF2 Signaling Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy

Rationale: Cytokines such as TNFα have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). While TNFα can elicit different cellular responses including survival or death, the mechanisms underlying these divergent outcomes in the heart remains cryptic. The E3 ubiquitin ligase TRAF2 provides a critical signaling platform for K63 - linked ubiquitination of RIPK1, crucial for NF-kB activation by TNFα. Whether alterations in TNFα-TRAF2 signaling underlie the cardiotoxic effects of DOX, remains poorly understood. Results: In contrast to vehicle treated mice, severe ultrastructural defects including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFa levels were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFa in DOX cardiotoxicity, we discovered that in the absence of DOX, NF-kB was readily activated by TNFa. However, TNFα -mediated NF-kB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked poly-ubiquitination of RIPK1, attributed to the proteasomal degradation of TRAF2. Further, TRAF2 protein expression was markedly reduced in hearts of cancer patients treated with DOX. Impaired TRAF2 signaling resulted in the activation of Bnip3 and mitochondrial perturbations, including disrupted bioenergetics, loss of membrane potential and permeability transition pore opening. We further established that the reciprocal actions of the ubiquitinating and de-ubiquitinating enzymes c-IAP1 and USP19 respectively regulated the proteasomal degradation of TRAF2 in DOX treated cardiac myocytes. Importantly, an E3 ligase mutant of c-IAP1(c-IAP1 H588A) or gain of function of USP19, prevented proteasomal degradation of TRAF2 and DOX -induced cell death. Further, wild type TRAF2 but not a RING finger mutant defective for K63 ubiquitination of RIPK1, restored NF-kB signaling and suppressed DOX-induced cardiac cell death. Finally, cardiomyocyte-restricted expression of TRAF2 (AAV9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. Conclusions: Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX, sensitized cardiac myocytes to TNFa mediated necrotic cell death.

Wearable Surface Deformation Myography (sDMG) System for Recognition of Locomotion Modes.

This study designs a wearable sensing system for locomotion mode recognition using lower-limb skin surface curvature deformation caused by the morphological changes of musculotendinous complexes and soft tissues. Flexible bending sensors are embedded into stretch pants, enabling curvature deformations of specific skin segments above lower-limb muscle groups to be captured in a noncontact manner. To evaluate the performance of this system, we conducted experiments on eight able-bodied subjects completing seven common locomotive activities, including walking, running, ramp ascending/descending, stair ascending/descending, and standing. The system measured seven channels of deformation signals from two cross-sections on the shank and the thigh. The collected signals were distinguishable across different locomotion modes and exhibited consistency when monitoring steps. Using selected time-domain features and a linear discriminant analysis (LDA) classifier enabled the proposed system to continuously recognize locomotion modes with an average accuracy of 96.5%. Furthermore, the system maintains recognition performance with 95.7% accuracy even after removing and reapplying the sensors. Finally, we conducted comparison experiments to analyze how window length, feature selection, and the number of channels affect recognition performance, providing insights for optimization. We believe that this novel signal platform holds great potential as a valuable supplementary tool in wearable human motion detection, enriching the information diversity for motion analysis, and enabling new possibilities for further advancements and applications in fields including biomedical engineering, textiles, and computer graphics.

Flexible and multi-functional three-dimensional scaffold based on enokitake-like Au nanowires for real-time monitoring of endothelial mechanotransduction

Endothelial cells are sensitive to mechanical force and can convert it into biochemical signals to trigger mechano-chemo-transduction. Although conventional techniques have been used to investigate the subsequent modifications of cellular expression after mechanical stimulation, the in situ and real-time acquiring the transient biochemical information during mechanotransduction process remains an enormous challenge. In this work, we develop a flexible and multi-functional three-dimensional conductive scaffold that integrates cell growth, mechanical stimulation, and electrochemical sensing by in situ growth of enokitake-like Au nanowires on a three-dimensional porous polydimethylsiloxane substrate. The conductive scaffold possesses stable and desirable electrochemical sensing performance toward nitric oxide under mechanical deformation. The prepared e-AuNWs/CC/PDMS scaffold exhibits a good electrocatalytic ability to NO with a linear range from 2.5 nM to 13.95 μM and a detection limit of 8 nM. Owing to the excellent cellular compatibility, endothelial cells can be cultured directly on the scaffold and the real-time inducing and recording of nitric oxide secretion under physiological and pathological conditions were achieved. This work renders a reliable sensing platform for real-time monitoring cytomechanical signaling during endothelial mechanotransduction and is expected to promote other related biological investigations based on three-dimensional cell culture.

The core spindle pole body scaffold Ppc89 links the pericentrin orthologue Pcp1 to the fission yeast spindle pole body via an evolutionarily conserved interface.

Centrosomes and spindle pole bodies (SPBs) are important for mitotic spindle formation and serve as cellular signaling platforms. Although centrosomes and SPBs differ in morphology, many mechanistic insights into centrosome function have been gleaned from SPB studies. In the fission yeast Schizosaccharomyces pombe, the α-helical protein Ppc89, identified based on its interaction with the septation initiation network scaffold Sid4, comprises the SPB core. High-resolution imaging has suggested that SPB proteins assemble on the Ppc89 core during SPB duplication, but such interactions are undefined. Here, we define a connection between Ppc89 and the essential pericentrin Pcp1. Specifically, we found that a predicted third helix within Ppc89 binds the Pcp1 pericentrin-AKAP450 centrosomal targeting (PACT) domain complexed with calmodulin. Ppc89 helix 3 contains similarity to present in the N-terminus of Cep57 (PINC) motifs found in the centrosomal proteins fly SAS-6 and human Cep57 and also to the S. cerevisiae SPB protein Spc42. These motifs bind pericentrin-calmodulin complexes and AlphaFold2 models suggest a homologous complex assembles in all four organisms. Mutational analysis of the S. pombe complex supports the importance of Ppc89-Pcp1 binding interface in vivo. Our studies provide insight into the core architecture of the S. pombe SPB and suggest an evolutionarily conserved mechanism of scaffolding pericentrin-calmodulin complexes for mitotic spindle formation.

Antimicrobial cetylpyridinium chloride suppresses mast cell function by targeting tyrosine phosphorylation of Syk kinase.

Cetylpyridinium chloride (CPC) is a quaternary ammonium antimicrobial used in numerous personal care products, human food, cosmetic products, and cleaning solutions. Yet, there is minimal published data on CPC effects on eukaryotes, immune signaling, and human health. Previously, we showed that low-micromolar CPC inhibits rat mast cell function by inhibiting antigen (Ag)-stimulated Ca 2+ mobilization, microtubule polymerization, and degranulation. In this study, we extend the findings to human mast cells (LAD2) and present data indicating that CPC's mechanism of action centers on its positively-charged quaternary nitrogen in its pyridinium headgroup. CPC's inhibitory effect is independent of signaling platform receptor architecture. Tyrosine phosphorylation events are a trigger of Ca 2+ mobilization necessary for degranulation. CPC inhibits global tyrosine phosphorylation in Ag-stimulated mast cells. Specifically, CPC inhibits tyrosine phosphorylation of specific key players Syk kinase and LAT, a substrate of Syk. In contrast, CPC does not affect Lyn kinase phosphorylation. Thus, CPC's root mechanism is electrostatic disruption of particular tyrosine phosphorylation events essential for signaling. This work outlines the biochemical mechanisms underlying the effects of CPC on immune signaling and allows the prediction of CPC effects on cell types, like T cells, that share similar signaling elements.

The delayed kinetics of Myddosome formation explains why amyloid-beta aggregates trigger Toll-like receptor 4 less efficiently than lipopolysaccharide

The Myddosome is a key innate immune signalling platform. It forms at the cell surface and contains MyD88 and IRAK proteins which ultimately coordinate the production of pro-inflammatory cytokines. Toll-like receptor 4 (TLR4) signals via the Myddosome when triggered by lipopolysaccharide (LPS) or amyloid-beta (Aβ) aggregates but the magnitude and time duration of the response are very different for reasons that are unclear. Here, we followed the formation of Myddosomes in live macrophages using local delivery of TLR4 agonist to the cell surface and visualisation with 3D rapid light sheet imaging. This was complemented by super-resolution imaging of Myddosomes in fixed macrophages to determine the size of the signalling complex at different times after triggering. Myddosomes formed more rapidly after LPS than in response to sonicated Aβ 1–42 fibrils (80 vs 372 s). The mean lifetimes of the Myddosomes were also shorter when triggered by LPS compared to sonicated Aβ fibrils (170 and 220 s), respectively. In both cases, a range of Myddosome of different sizes (50–500 nm) were formed. In particular, small round Myddosomes around 100 nm in size formed at early time points, then reduced in proportion over time. Collectively, our data suggest that compared to LPS the multivalency of Aβ fibrils leads to the formation of larger Myddosomes which form more slowly and, due to their size, take longer to disassemble. This explains why sonicated Aβ fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates.

Artificial and Natural Sweeteners Biased T1R2/T1R3 Taste Receptors Transactivate Glycosylated Receptors on Cancer Cells to Induce Epithelial-Mesenchymal Transition of Metastatic Phenotype.

Understanding the role of biased taste T1R2/T1R3 G protein-coupled receptors (GPCR) agonists on glycosylated receptor signaling may provide insights into the opposing effects mediated by artificial and natural sweeteners, particularly in cancer and metastasis. Sweetener-taste GPCRs can be activated by several active states involving either biased agonism, functional selectivity, or ligand-directed signaling. However, there are increasing arrays of sweetener ligands with different degrees of allosteric biased modulation that can vary dramatically in binding- and signaling-specific manners. Here, emerging evidence proposes the involvement of taste GPCRs in a biased GPCR signaling crosstalk involving matrix metalloproteinase-9 (MMP-9) and neuraminidase-1 (Neu-1) activating glycosylated receptors by modifying sialic acids. The findings revealed that most natural and artificial sweeteners significantly activate Neu-1 sialidase in a dose-dependent fashion in RAW-Blue and PANC-1 cells. To confirm this biased GPCR signaling crosstalk, BIM-23127 (neuromedin B receptor inhibitor, MMP-9i (specific MMP-9 inhibitor), and oseltamivir phosphate (specific Neu-1 inhibitor) significantly block sweetener agonist-induced Neu-1 sialidase activity. To assess the effect of artificial and natural sweeteners on the key survival pathways critical for pancreatic cancer progression, we analyzed the expression of epithelial-mesenchymal markers, CD24, ADLH-1, E-cadherin, and N-cadherin in PANC-1 cells, and assess the cellular migration invasiveness in a scratch wound closure assay, and the tunneling nanotubes (TNTs) in staging the migratory intercellular communication. The artificial and natural sweeteners induced metastatic phenotype of PANC-1 pancreatic cancer cells to promote migratory intercellular communication and invasion. The sweeteners also induced the downstream NFκB activation using the secretory alkaline phosphatase (SEAP) assay. These findings elucidate a novel taste T1R2/T1R3 GPCR functional selectivity of a signaling platform in which sweeteners activate downstream signaling, contributing to tumorigenesis and metastasis via a proposed NFκB-induced epigenetic reprogramming modeling.

The delayed kinetics of Myddosome formation explains why amyloid-beta aggregates trigger Toll-like receptor 4 less efficiently than lipopolysaccharide.

The Myddosome is a key innate immune signalling platform. It forms at the cell surface and contains MyD88 and IRAK proteins which ultimately coordinate the production of pro-inflammatory cytokines. Toll-like receptor 4 (TLR4) signals via the Myddosome when triggered by lipopolysaccharide (LPS) or amyloid-beta (Aβ) aggregates but the magnitude and time duration of the response are very different for reasons that are unclear. Here, we followed the formation of Myddosomes in live macrophages using local delivery of TLR4 agonist to the cell surface and visualisation with 3D rapid light sheet imaging. This was complemented by super-resolution imaging of Myddosomes in fixed macrophages to determine the size of the signalling complex at different times after triggering. Myddosomes formed more rapidly after LPS than in response to sonicated Aβ 1-42 fibrils (80 vs 372 s). The mean lifetimes of the Myddosomes were also shorter when triggered by LPS compared to sonicated Aβ fibrils (170 and 220 s), respectively. In both cases, a range of Myddosome of different sizes (50-500 nm) were formed. In particular, small round Myddosomes around 100 nm in size formed at early time points, then reduced in proportion over time. Collectively, our data suggest that compared to LPS the multivalency of Aβ fibrils leads to the formation of larger Myddosomes which form more slowly and, due to their size, take longer to disassemble. This explains why sonicated Aβ fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates.