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Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular receptor for muramyl dipeptide derived from the intestinal microbiota. Loss-of-function mutations in Nod2 are associated with the development of Crohn's disease, suggesting that NOD2 signaling plays critical roles in the maintenance of intestinal immune homeostasis. Although NOD2 activation prevents the development of short-term experimental colitis, it remains unknown whether the sensitivity to long-term experimental colitis is influenced by NOD2. In this study, we explored the roles played by NOD2 in the development of long-term adoptive transfer colitis. Unexpectedly, we found that Rag1-/-Nod2-/- mice were more resistant to adoptive transfer colitis than Rag1-/- mice and had reduced proinflammatory cytokine responses and enhanced accumulation of regulatory T cells (Tregs) expressing forkhead box P3 in the colonic mucosa. Prevention of colitis in Rag1-/-Nod2-/- mice was mediated by TGF-β1 because neutralization of TGF-β1 resulted in the development of more severe colitis due to reduced accumulation of Tregs. Such paradoxical Treg responses in the absence of NOD2 could explain why Nod2 mutations in humans are not sufficient to cause Crohn's disease.

Objective Nod-like receptor pyrin domain containing 3 ( NLRP3) gene encodes an intracellular receptor whose dysregulation in systemic lupus erythematosus (SLE) has been reported in multiple studies. Activation of NLRP3 inflammasome leads to the induction of inflammatory response via cleaving and producing of specific cytokines. In the present study, we assessed the possible association between three functional polymorphisms in this gene and SLE risk in the Iranian population. These variants include two gain of function (rs4612666 and rs10754558) and one loss of function (rs6672995) which are correlated with modulation of expression of NLRP3. Methods A case-control study involving 110 SLE patients and 116 control subjects was undertaken to estimate the frequency of rs4612666, rs10754558, and rs6672995 genotypes using real-time PCR high resolution melting method (HRM). Results Our findings revealed significant associations between GG genotype and G allele of rs10754558 with increased risk of SLE (OR for GG genotype= 2.82; 95%CI [1.45–5.46]/OR for G allele= 1.97; 95%CI [1.36–2.87]). Although, no significant associations were recognized between allele and genotype frequencies of rs4612666 and rs6672995 polymorphisms with SLE risk ( P > 0.05). Also, our analysis revealed that the C allele in rs4612666 and G allele in rs10754558 was correlated with the severity of disease activity (P Conclusion This study demonstrates a substantial association between NLRP3 polymorphisms with increased risk, clinical symptoms, and the severity of disease activity of SLE.

Bacterial-derived RNA and DNA can function as ligands for intracellular receptor activation and induce downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory RNA and DNA by pathogens such as Staphylococcus aureus and their delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. S. aureus produce membrane-bound, spherical, nano-sized, MVs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. Here we show that S. aureus secretes RNA and DNA molecules that are mostly protected from degradation by their association with MVs. Importantly, we demonstrate that MVs can be delivered into cultured macrophage cells and subsequently stimulate a potent IFN-β response in recipient cells via activation of endosomal Toll-like receptors. These findings advance our understanding of the mechanisms by which bacterial nucleic acids traffic extracellularly to trigger the modulation of host immune responses.

Sigma non-opioid intracellular receptor 1 (sigma-1 receptor), a non-opioid transmembrane protein, is located on cellular mitochondrial membranes and endoplasmic reticulum. Current research has demonstrated that sigma-1 receptor is related to human degenerative diseases. This study is focused on the effects of sigma-1 receptor on the pathophysiological process of diminished ovarian reserve (DOR) and granulosa cells (GCs) apoptosis. Sigma-1 receptor concentration in follicular fluid (FF) and serum were negatively correlated with basal follicle-stimulating hormone (FSH) and positively correlated with anti-mullerian hormone (AMH), antral follicle count (AFC). Sigma-1 receptor reduction in GCs was accompanied by endoplasmic reticulum stress (ERS)-mediated apoptosis in women with DOR. Plasmid transfection was used to establish SIGMAR1-overexpressed and SIGMAR1-knockdown human granulosa-like tumor (KGN) cell and thapsigargin (TG) was used to induce ERS KGN cells. We found that KGN cells treated with endogenous sigma-1 receptor ligand dehydroepiandrosterone (DHEA) and sigma-1 receptor agonist PRE-084 showed similar biological effects to SIGMAR1-overexpressed KGN cells and opposite effects to SIGMAR1-knockdown KGN cells. DHEA may improve DOR patients' pregnancy outcomes by upregulating sigma-1 receptor and downregulating ERS-mediated apoptotic genes in GCs. Thus, sigma-1 receptor may be a potential ovarian reserve biomarker, and ligand-mediated sigma-1 receptor activation could be a future approach for DOR therapy.

Background Juvenile hormones (JHs) are a class of sesquiterpenoids that play a pivotal role in insect growth and reproduction. Synthetic JH agonists (JHAs), including pyriproxyfen, have been widely used as insecticides to control agricultural pests and disease vectors. The antimetamorphic action of JHAs is mediated by their intracellular receptor, the heterodimer of Methoprene-tolerant (Met) and Taiman (Tai) proteins. Although a range of bioassay systems has been developed to detect the activity of JHAs, each of these systems has its own drawback(s), such as poor reproducibility, the use of radioactive ligands or the effect of endogenous JH-signaling factors. Results To address these issues, we constructed a new luciferase reporter assay for JHAs in mammalian HEK293T cells transiently transfected with the Drosophila Met and Tai genes. This reporter system gave highly reproducible results and showed nanomolar sensitivity to natural JHs. We then applied this reporter system to a structure-activity relationship (SAR) analysis of 14 natural and synthetic JHAs, leading to identification of the ligand structural factors important for the transcription-inducing activity. Conclusion Because this reporter system is not affected by the signaling cascade downstream of the JH receptors, it is suitable for evaluating the intrinsic activity of JHAs. The SAR results obtained in this study therefore provide invaluable information on the rational design of novel JHA insecticides.

Brain inflammation generally accompanies and accelerates neurodegeneration. Here we report a microglial mechanism in which polyglutamine binding protein 1 (PQBP1) senses extrinsic tau 3R/4R proteins by direct interaction and triggers an innate immune response by activating a cyclic GMP-AMP synthase (cGAS)-Stimulator of interferon genes (STING) pathway. Tamoxifen-inducible and microglia-specific depletion of PQBP1 in primary culture in vitro and mouse brain in vivo shows that PQBP1 is essential for sensing-tau to induce nuclear translocation of nuclear factor κB (NFκB), NFκB-dependent transcription of inflammation genes, brain inflammation in vivo, and eventually mouse cognitive impairment. Collectively, PQBP1 is an intracellular receptor in the cGAS-STING pathway not only for cDNA of human immunodeficiency virus (HIV) but also for the transmissible neurodegenerative disease protein tau. This study characterises a mechanism of brain inflammation that is common to virus infection and neurodegenerative disorders., Brain inflammation generally accelerates neurodegeneration but the mechanisms of this are not fully characterised. Here the authors show that PQBP1 in microglia is important for sensing extrinsic Tau 3 R/4 R proteins and triggers an innate immune response through cGAS and STING resulting in cognitive impairment.

Different families of auxiliary subunits regulate the function and trafficking of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the central nervous system. While a facilitatory role of auxiliary subunits in ER export and forward trafficking of newly synthesized AMPA receptors is firmly established, it is unclear whether auxiliary subunits also control endosomal receptor turnover in dendrites. Here, we manipulated the composition of AMPA receptor complexes in cultured hippocampal neurons by overexpression of two auxiliary subunits, transmembrane AMPAR regulatory protein (TARP) γ-8 or cysteine knot AMPAR-modulating protein (CKAMP) 44a, and monitored dendritic receptor cycling in live-cell imaging experiments. Receptor surface delivery was assayed using a modified AMPA receptor subunit carrying the pH-dependent fluorophore superecliptic pHluorin (SEP-GluA1), which regains its fluorescence during receptor exocytosis, when transiting from the acidic lumen of transport organelles to the neutral extracellular medium. Strikingly, we observed a dramatic reduction in the spontaneous fusion rate of AMPA receptor-containing organelles in neurons overexpressing either type of auxiliary subunit. An analysis of intracellular receptor distribution also revealed a decreased receptor pool in dendritic recycling endosomes, suggesting that incorporation of TARPγ-8 or CKAMP44a in receptor complexes generally diminishes cycling through the endosomal compartment. To directly analyze dendritic receptor turnover, we also generated a new reporter by N-terminal fusion of a self-labeling HaloTag to an AMPA receptor subunit (HaloTag-GluA1), which allows for selective, irreversible staining of surface receptors. Pulse chase-experiments with HaloTag-GluA1 indeed demonstrated that overexpression of TARPγ-8 or CKAMP44a reduces the constitutive internalization rate of surface receptors at extrasynaptic but not synaptic sites. Thus, our data point to a yet unrecognized regulatory function of TARPγ-8 and CKAMP44a, by which these structurally unrelated auxiliary subunits delay local recycling and increase surface lifetime of extrasynaptic AMPA receptors.

Targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), especially severe cases, are currently lacking. As macrophages have unique effector functions as a first-line defense against invading pathogens, we genetically armed human macrophages with chimeric antigen receptors (CARs) to reprogram their phagocytic activity against SARS-CoV-2. After investigation of CAR constructs with different intracellular receptor domains, we found that although cytosolic domains from MERTK (CARMERTK) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcRγ and CD3ζ did, these CARs all mediated similar SARS-CoV-2 clearance in vitro. Notably, we showed that CARMERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CARMERTK drives an ‘immunologically silent’ scavenger effect in macrophages and pave the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation.

Cortisol is a critical neuroendocrine regulator of the stress response in fish. Cortisol practically affects all tissues by interacting with an intracellular receptor and modulating target gene expression. However, cortisol also interacts with components of the plasma membrane in a nongenomic process that activates rapid signaling. Until now, the implication of this novel cortisol signaling for the global transcriptional response has not been explored. In the present work, we evaluated the effects of the membrane-initiated actions of cortisol on the in vivo transcriptome of rainbow trout ( Oncorhynchus mykiss) skeletal muscle. RNA-Seq analyses were performed to examine the transcriptomic changes in rainbow trout stimulated by physiological concentrations of cortisol and cortisol coupled with bovine serum albumin (cortisol-BSA), a membrane-impermeable analog of cortisol. A total of 660 million paired-ends reads were generated. Reads mapped onto the reference genome revealed that 1,737; 897; and 1,012 transcripts were differentially expressed after 1, 3, and 9 h of cortisol-BSA treatment, respectively. Gene Ontology analysis showed that this novel action of cortisol modulates several biological processes, such as mRNA processing, ubiquitin-dependent protein catabolic processes, and transcription regulation. In addition, a KEGG analysis revealed that focal adhesion was the main signaling pathway that was upregulated at all the times tested. Taking these results together, we propose that the membrane-initiated cortisol action contributes significantly in the regulation of stress-mediated gene expression.

This study focuses on the biological prospects of oxygen-depleted calix[4]arene ligands on a protein target. Because of their extensive medical relevance, the oxygen-depleted bis(piperdine) (BPD) and bis(pyrazole) (BPZ) ligands were synthesized and characterized by NMR and mass spectrometry. Furthermore, molecular docking followed by molecular dynamics simulation was utilized to understand the behavior of ligands on selection of sigma non-opiod intracellular receptor (SigNOR) as a suitable protein target. The simulations were carried out by three level of complexity: (1) Apo SigNOR, (2) BPD: SigNOR, and (3) BPZ: SigNOR. The three complex systems were subjected to stability check before detail analysis. From the results of the estimation of binding free energy, it follows that both ligands possess the same free energy of binding which, in turn, suggests their similar role; however, energy components such as Van der waal and electrostatic potential recommend BPZ were identified as a competitive drug on SigNOR. In addition, temporal distribution of the clusters suggests that scattering of the cluster’s popularity is a measure of fast structural transitions in both complexes. Current study utilizes modern approach to synthesis, characterization, and simulation of our ligands. This study appropriately highlights the effect of our ligands on SigNOR Protein, which might further be extended to potential in vitro and in vivo bioassay.

Lipopolysaccharide (LPS) sensing in the cytosol by the noncanonical inflammasome leads to pyroptosis and NLRP3 inflammasome activation. This mechanism may be more critical for sepsis development than recognition of LPS by Toll-like receptor 4. LPS is directly binding to its intracellular receptor caspase-4/5/11, mediated by outer membrane vesicles and guanylate-binding proteins that deliver LPS to the cytosol and mediate access of caspases to LPS. Caspase-11-dependent cleavage of gasdermin D is discussed as a link between LPS-induced activation of caspases and pyroptosis or NLRP3 inflammasome activation. Finally, we highlight recently described inhibitors of cytosolic LPS-triggered noncanonical inflammasome activation that might be considered as potential drugs for the treatment of sepsis.

The TSH receptor (TSHR) is the major autoantigen in Graves’ disease (GD). Bioinformatic analyses predict the existence of several human TSHR isoforms from alternative splicing, which can lead to the coexpression of multiple receptor forms. The most abundant of these is TSHRv1.3. In silico modeling of TSHRv1.3 demonstrated the structural integrity of this truncated receptor isoform and its potential binding of TSH. Tissue profiling revealed wide expression of TSHRv1.3, with a predominant presence in thyroid, bone marrow, thymus, and adipose tissue. To gain insight into the role of this v1.3 receptor isoform in thyroid pathophysiology, we cloned the entire open reading frame into a mammalian expression vector. Immunoprecipitation studies demonstrated that both TSHR-stimulating antibody and human TSH could bind v1.3. Furthermore, TSHRv1.3 inhibited the stimulatory effect of TSH and TSHR-Ab MS-1 antibody on TSHR-induced cAMP generation in a dose-dependent manner. To confirm the antigenicity of v1.3, we used a peptide ELISA against two different epitopes. Of 13 GD samples, 11 (84.6%) were positive for a carboxy terminal peptide and 10 (76.9%) were positive with a junction region peptide. To demonstrate that intracellular v1.3 could serve as an autoantigen and modulate disease, we used double-transfected Chinese hamster ovary cells that expressed both green fluorescent protein (GFP)-tagged TSHRv1.3 and full-length TSHR. We then induced cell stress and apoptosis using a TSHR monoclonal antibody and observed the culture supernatant contained v1.3-GFP protein, demonstrating the release of the intracellular receptor variant by this mechanism.

Epidemiological studies correlate sun or UV light exposure with a lower incidence of a variety of malignancies, including breast, colon and prostate cancer. The biologically active 1,25(OH)2D3 and its analogs have been shown to have antiproliferative and differentiating effects in a variety of malignant and non-malignant cells. The effects of 1,25(OH)2D3 are mediated by the binding of calcitriol to a specific intracellular receptor, vitamin D receptor [VDR]. The aim of this study is to review the literature concerning the role of 1,25(OH)2D3 and its analogs in squamous carcinoma cell lines of the head and neck (SCCHN).

The macrolide antibiotics, FK 506 and rapamycin, have been reported to be potent immunosuppressive drugs. Both FK506 and rapamycin bind to the immunophilin macrophilin-12. Rapamycin, however, acts on T cells by a different mode of action which does not affect the transcription of lymphokines. The anti-inflammatory effects of FK 506 and rapamycin have been tested in mouse models of irritant dermatitis after topical application, and in allergic contact dermatitis after topical or oral application, using mice and domestic pigs. In irritant dermatitis, both FK 506 and rapamycin exerted significant, but moderate topical activity at concentrations of 0.4-3.6%. In allergic contact dermatitis, topical FK 506 proved to be highly active at concentrations of 0.01-0.1% and even superior to corticosteroids. In contrast, rapamycin was inactive. After oral treatment of mice, FK506 was significantly active (2 × 30mg/kg/ day) while rapamycin was inactive up to the highest dose tested (2 × 90 mg/kg/ day). These results indicate that, despite the chemical similarity of FK 506 and rapamycin and their common intracellular receptor, there are different fundamental pharmacological properties of FK 506 and rapamycin, which may reflect their different modes of action.

NOD1 is an intracellular receptor that, when activated, induces gene expression of pro-inflammatory factors promoting macrophages and neutrophils recruitment at the infection site. However, iE-DAP, the dipeptide agonist that promotes this receptor's activation, cannot permeate cell membranes. To develop a nanocarrier capable of achieving a high and prolonged activation over time, iE-DAP was encapsulated in nanoparticles (NPs) made of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The physicochemical properties, colloidal stability, encapsulation efficiency, and cellular uptake of iE-DAP-loaded PHVB NPs were analyzed. Results evidenced that physicochemical properties of iE-DAP-loaded NPs remained stable over time, and NPs were efficiently internalized into cells, a process that depends on time and concentration. Moreover, our results showed that NPs elicited a controlled cargo release in vitro, and the encapsulated agonist response was higher than its free form, suggesting the possibility of activating intracellular receptors triggering an immune response through the release of NOD1 agonist.

Supplemental Digital Content is available in the text., OBJECTIVES: Critical illness is characterized by increased serum cortisol concentrations and bioavailability resulting from the activation of the hypothalamic-pituitary-adrenal axis, which constitutes an essential part of the stress response. The actions of glucocorticoids are mediated by a ubiquitous intracellular receptor protein, the glucocorticoid receptor. So far, data on coronavirus disease 2019 and glucocorticoid receptor alpha expression are lacking. DESIGN: Prospective observational study. SETTING: One academic multidisciplinary ICU. SUBJECTS: Twenty-six adult coronavirus disease 2019 patients; 33 adult noncoronavirus disease 2019 patients, matched for age, sex, and disease severity, constituted the control group. All patients were steroid-free. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Glucocorticoid receptor alpha, glucocorticoid-inducible leucine zipper expression, and serum cortisol were measured on ICU admission. In coronavirus disease 2019 patients, glucocorticoid receptor alpha and glucocorticoid-inducible leucine zipper messenger RNA expression were upregulated (4.7-fold, p < 0.01 and 14-fold, p < 0.0001, respectively), and cortisol was higher (20.3 vs 14.3 μg/dL, p < 0.01) compared with the control group. CONCLUSIONS: ICU coronavirus disease 2019 patients showed upregulated glucocorticoid receptor alpha and glucocorticoid-inducible leucine zipper expression, along with cortisol levels, compared with ICU noncoronavirus disease 2019 patients. Thus, on ICU admission, critical coronavirus disease 2019 appears to be associated with hypercortisolemia, and increased synthesis of glucocorticoid receptor alpha and induced proteins.

To support their innate immune and scavenging functions in the brain, microglia are equipped with Toll-like receptors (TLRs), including the intracellular receptor TLR9, which is activated by microbial CpG-rich DNA. Macropinocytosis is an abundant and inducible pathway in microglia for fluid-phase uptake and ingestion of microbes and cell debris. TLR9 signaling has been ascribed to endolysosomes, particularly lysosomes, which it accesses through direct transport or via internalization from the surface. Here, TLR9 and exogenous CpG-DNA are localized during uptake into fluid-filled macropinosomes, upon upregulated macropinocytosis, where acidic and proteolytic environments support MyD88-induced signaling. Macropinosomes represent an abundant pathway for endolysosomal traffic of TLR9 but are also a much more exposed site for nucleic acid activation of the receptor with a risk of excessive inflammation. To constrain TLR9 inflammation, macropinosomes also house the TLR9 co-receptor LRP1 and regulators Rab8a and PI3Kγ which augment Akt signaling and favor anti-inflammatory cytokine production. Macropinosomes and their inflammatory regulators are therefore important components of TLR9 pathways in microglia that are poised for surveillance and protection in the CNS.

The mineralocorticoid receptor (MR) mediates the action of two adrenal steroid hormones, aldosterone, and cortisol, in many tissues in response to normal physiological change and in response to stress, adaption, or disease states. Aldosterone is a key regulator of cardiovascular homeostasis, and cortisol mediates adaptive responses to stress, both centrally and in the periphery. The MR is a large multidomain intracellular receptor that once activated triggers multiple cytoplasmic signaling pathways and in the nucleus directly regulates target gene expression to change cellular responses. Human MR gene mutations cause forms of pseudohypoaldosteronism-type-1 with prominent renal dysfunction, abnormalities in HPA axis activity, and cognitive behavior and have recently been associated with autism. MR mutations modeled in the mouse and fish are defining tissue-specific functions and molecular mechanisms related to normal function and disease. Future research will define new roles for the MR in the body and provide further detail of MR-activated cellular responses.

Synthetic receptors control cell behavior in response to environmental stimuli for applications in basic research and cell therapy. However, the integration of synthetic receptors in unexplored contexts is cumbersome, especially for nonspecialist laboratories. Here, I provide a detailed protocol on how to use receptors of the generalized extracellular molecule sensor (GEMS) platform. GEMS is a modular receptor system that can be adapted to sense molecules of choice by using affinity domains that dimerize in response to the target. GEMS consist of an erythropoietin receptor scaffold that has been mutated to no longer bind to erythropoietin. N-terminal fusions with affinity domains, such as single chain variable fragments (scFvs), that bind to two epitopes on the same target activate the receptor. The intracellular receptor domain can be chosen from several signal transduction domains of single-pass transmembrane receptors to activate endogenous signaling pathways. As of now, GEMS have been used for sensing prostate specific antigen (PSA), the synthetic azo dye RR120, caffeine, nicotine, rapamycin, the SunTag peptide, and a de novo designed protein displaying two viral epitopes. The tested intracellular domains were derived from FGFR1, IL-6RB, and VEGFR2, and were used to drive transgene expression from reporter plasmids responsive to the endogenous transcription factors STAT3, NFAT, NF-κB, and a synthetic transcription factor activated by the MAPK pathway. In this protocol, I focus on transient transfections of HEK293T cells and include several general notes about cell handling. While the described methods are optimized for experiments with GEMS, most of the described techniques are general procedures to set up synthetic biology experiments in mammalian cell culture. I outline how to generate stable cell lines and share tips on how to adapt GEMS for new ligands. The main objective of this protocol is to make the GEMS technology accessible also to nonspecialist laboratories to facilitate the use of synthetic receptors in new research contexts.

Endocannabinoid signaling depends upon the CB1 and CB2 cannabinoid receptors, their endogenous ligands anandamide and 2-arachidonoylglycerol, and intracellular proteins that mediate responses via the C-terminal and other intracellular receptor domains. The CB1 receptor regulates and is regulated by associated G proteins predominantly of the Gi/o subtypes, β-arrestins 1 and 2, and the cannabinoid receptor-interacting protein 1a (CRIP1a). Evidence for a physiological role for CRIP1a is emerging as data regarding the cellular localization and function of CRIP1a are generated. Here we summarize the neuronal distribution and role of CRIP1a in endocannabinoid signaling, as well as discuss investigations linking CRIP1a to development, vision and hearing sensory systems, hippocampus and seizure regulation, and psychiatric disorders including schizophrenia. We also examine the genetic and epigenetic association of CRIP1a within a variety of cancer subtypes. This review provides evidence upon which to base future investigations on the function of CRIP1a in health and disease.

Targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), especially severe cases, are currently lacking. As macrophages have unique effector functions as a first-line defense against invading pathogens, we genetically armed human macrophages with chimeric antigen receptors (CARs) to reprogram their phagocytic activity against SARS-CoV-2. After investigation of CAR constructs with different intracellular receptor domains, we found that although cytosolic domains from MERTK (CARMERTK) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcRγ and CD3ζ did, these CARs all mediated similar SARS-CoV-2 clearance in vitro. Notably, we showed that CARMERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CARMERTK drives an ‘immunologically silent’ scavenger effect in macrophages and pave the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation.

Amyotrophic lateral sclerosis (ALS) is an adult disease causing a progressive loss of upper and lower motoneurons, muscle paralysis and early death. ALS has a poor prognosis of 3-5 years after diagnosis with no effective cure. The aetiopathogenic mechanisms involved include glutamate excitotoxicity, oxidative stress, protein misfolding, mitochondrial alterations, disrupted axonal transport and inflammation. Sigma non-opioid intracellular receptor 1 (sigma 1 receptor) is a protein expressed in motoneurons, mainly found in the endoplasmic reticulum (ER) on the mitochondria-associated ER membrane (MAM) or in close contact with cholinergic postsynaptic sites. MAMs are sites that allow the assembly of several complexes implicated in essential survival cell functions. The sigma 1 receptor modulates essential mechanisms for motoneuron survival including excitotoxicity, calcium homeostasis, ER stress and mitochondrial dysfunction. This review updates sigma 1 receptor mechanisms and its alterations in ALS, focusing on MAM modulation, which may constitute a novel target for therapeutic strategies. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.6/issuetoc.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is an intracellular receptor for polysialic acid. MARCKS supports development, synaptic plasticity, and regeneration after injury. MARCKS binds with its functionally essential effector domain (ED) to polysialic acid. A 25-mer peptide comprising the ED of MARCKS stimulates neuritogenesis of primary hippocampal neurons after addition to the culture. This motivated us to investigate whether ED peptide has similar effects in spinal cord injury. ED peptide supported recovery and regrowth of monoaminergic axons in female, but not in male mice. Sex-specific differences in response to ED peptide application also occurred in cultured neurons. In female but not male neurons, the ED peptide enhanced neurite outgrowth that could be suppressed by inhibitors of the estrogen receptors α and β, fibroblast growth factor receptor-1, protein kinase C, and matrix metalloproteinase 2. In addition, we observed female-specific elevation of phosphorylated MARCKS levels after ED peptide treatment. In male neurons, the ED peptide enhanced neuritogenesis in the presence of an androgen receptor inhibitor to the extent seen in ED peptide-treated female neurons. However, inhibition of androgen receptor did not lead to increased phosphorylation of MARCKS. These results provide insights into the functions of a novel compound contributing to gender-dependent regeneration.

Ligand-dependent oligomerization of receptor tyrosine kinases (RTKs) results in their activation through highly specific conformational changes in the extracellular and intracellular receptor domains. These conformational changes are unique for each RTK sub-family, limiting cross-activation between unrelated RTKs. The proto-oncogene MET receptor tyrosine kinase overcomes these structural constraints and phosphorylates unrelated RTKs in numerous cancer cell lines. The molecular basis for these interactions is unknown. We investigated the mechanism by which MET phosphorylates the human epidermal growth factor receptor-3 (HER3 or ERBB3), a catalytically impaired RTK whose phosphorylation by MET has been described as an essential component of drug resistance to inhibitors targeting EGFR and HER2. We find that in untransformed cells, HER3 is not phosphorylated by MET in response to ligand stimulation, but rather to increasing levels of MET expression, which results in MET activation in a ligand-independent manner. Phosphorylation of HER3 by its canonical dimerization partners, EGFR and HER2, is achieved by engaging an allosteric site on the HER3 kinase domain, but this site is not required when HER3 is phosphorylated by MET. We also observe that HER3 preferentially interacts with MET during its maturation along the secretory pathway, before MET is post-translationally processed by cleavage within its extracellular domain. This results in accumulation of phosphorylated HER3 in the Golgi apparatus. We further show that in addition to HER3, MET phosphorylates other RTKs in the Golgi, suggesting that this mechanism is not limited to HER3 phosphorylation. These data demonstrate a link between MET overexpression and its aberrant activation in the Golgi endomembranes and suggest that non-canonical interactions between MET and unrelated RTKs occur during maturation of receptors. Our study highlights a novel aspect of MET signaling in cancer that would not be accessible to inhibition by therapeutic antibodies.

Since it was first postulated by Wigglesworth in 1934, juvenile hormone (JH) is considered a status quo hormone in insects because it prevents metamorphosis that is initiated by the molting hormone 20-hydroxyecdysone (20E). During the last decade, significant advances have been made regarding JH signaling. First, the bHLH-PAS transcription factor Met/Gce was identified as the JH intracellular receptor. In the presence of JH, with the assistance of Hsp83, and through physical association with a bHLH-PAS transcriptional co-activator, Met/Gce enters the nucleus and binds to E-box-like motifs in promoter regions of JH primary-response genes for inducing gene expression. Second, the zinc finger transcription factor Kr-h1 was identified as the anti-metamorphic factor which transduces JH signaling. Via Kr-h1 binding sites, Kr-h1 represses expression of 20E primary-response genes (i.e. Br, E93 and E75) to prevent 20E-induced metamorphosis. Third, through the intracellular signaling, JH promotes different aspects of female reproduction. Nevertheless, this action varies greatly from species to species. Last, a hypothetical JH membrane receptor has been predicted to be either a GPCR or a tyrosine kinase receptor. In future, it will be a great challenge to understand how the JH intracellular receptor Met/Gce and the yet unidentified JH membrane receptor coordinate to regulate metamorphosis and reproduction in insects.

Purpose Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis – BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. Material and methods Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (Gαq/11, Gαs, Gαi) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor®488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. Results Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50–60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP-subtype coupling patterns. Detection of M3 interactions with endocytic trafficking proteins by PLA resulted in object sizes correlating with well-documented vesicle sizes of the endocytosis pathway. Conclusion PLA enabled highly specific detection of M3 receptor expression, demonstration of M3/GP differential coupling and intracellular M3 trafficking in human detrusor smooth muscle cells. This new approach minimized background fluorescence and antibody cross-reactions resulting from single antibody application, and enhanced specificity due to the use of two primary antibodies. Use of subcellular markers allowed visualization of subcellular receptor location. PLA/CLSM allows analyses of muscarinic “receptor – G protein – promiscuity” and intracellular trafficking even in bladder paraffin sections and may give new insights into the etiology and pathology of BPS/IC.

12 p.-5 fig.-2 tab.-1 graph. abst., Niemann-Pick C1 (NPC1) receptor is an intracellular protein located in late endosomes and lysosomes whose main function is to regulate intracellular cholesterol trafficking. Besides being postulated as necessary for the infection of highly pathogenic viruses in which the integrity of cholesterol transport is required, this protein also allows the entry of the Ebola virus (EBOV) into the host cells acting as an intracellular receptor. EBOV glycoprotein (EBOV-GP) interaction with NPC1 at the endosomal membrane triggers the release of the viral material into the host cell, starting the infective cycle. Disruption of the NPC1/EBOV-GP interaction could represent an attractive strategy for the development of drugs aimed at inhibiting viral entry and thus infection. Some of the today available EBOV inhibitors were proposed to interrupt this interaction, but molecular and structural details about their mode of action are still preliminary thus more efforts are needed to properly address these points. Here, we provide a critical discussion of the potential of NPC1 and its interaction with EBOV-GP as a therapeutic target for viral infections., The project leading to these results has received funding from “la Caixa” Banking Foundation under the project code LCF/PR/HR19/52160012. This research was partially supported through the Spanish Ministry of Science and Innovation (RTI2018-097305-R-I00), the Instituto de Investigación Carlos III, ISCIII, (FIS PI 1801007) and the European Commission Horizon 2020 Framework Programme Project VIRUSCAN FETPROACT-2016: 731868. M. M. holds a pre-doctoral FPU grant (FPU18/03493) from MICINN.

Receptor signalling events including those initiated following activation of cytokine and growth factor receptors and the well-characterised death receptors (tumour necrosis factor receptor, type 1, FasR and TRAIL-R1/2) are initiated at the cell surface through the recruitment and formation of intracellular multiprotein signalling complexes that activate divergent signalling pathways. Over the past decade, research studies reveal that many of these receptor-initiated signalling events involve the sequential proteolysis of specific receptors by membrane-bound proteases and the γ-secretase protease complexes. Proteolysis enables the liberation of soluble receptor ectodomains and the generation of intracellular receptor cytoplasmic domain fragments. The combined and sequential enzymatic activity has been defined as regulated intramembrane proteolysis and is now a fundamental signal transduction process involved in the termination or propagation of receptor signalling events. In this review, we discuss emerging evidence for a role of the γ-secretase protease complexes and regulated intramembrane proteolysis in cell- and immune-signalling pathways.

Discovery of conserved communication systems in the agriculturally important Paenibacillus bacteria. These systems are widespread, and some species encode more than 25 different peptide-receptor pairs., Paenibacillus polymyxa is an agriculturally important plant growth–promoting rhizobacterium. Many Paenibacillus species are known to be engaged in complex bacteria–bacteria and bacteria–host interactions, which in other species were shown to necessitate quorum sensing communication. However, to date, no quorum sensing systems have been described in Paenibacillus. Here, we show that the type strain P. polymyxa ATCC 842 encodes at least 16 peptide-based communication systems. Each of these systems is comprised of a pro-peptide that is secreted to the growth medium and processed to generate a mature short peptide. Each peptide has a cognate intracellular receptor of the RRNPP family, and we show that external addition of P. polymyxa communication peptides leads to reprogramming of the transcriptional response. We found that these quorum sensing systems are conserved across hundreds of species belonging to the Paenibacillaceae family, with some species encoding more than 25 different peptide-receptor pairs, representing a record number of quorum sensing systems encoded in a single genome.

Our earlier studies proposed a radically new idea suggesting interdependency between TNF-α/TNFR1 and IL-1β/IL-1R pathways in modulation of Staphylococcus aureus-induced CXCL8/CXCR1 axis. However, the effects of inhibition of cytokine receptor mobilization at intracellular level and surface TNFR1 and IL-1R shedding on S. aureus-induced CXCR1 expression have not been studied so far in peritoneal macrophages. This study aimed to investigate the role of inhibition of receptor mobilization from the intracellular pool (using brefeldin A) and surface receptor shedding (using TAPI-1) on CXCR1 expression during dual receptor (TNFR1 plus IL-1R) neutralization in peritoneal macrophages isolated from wild-type Swiss Albino mice. Release of superoxide anion, nitric oxide, and hydrogen peroxide was measured and cytokine production was done by ELISA. Expression of surface receptors (TNFR1, IL-1R, and CXCR1) and inflammatory mediators was studied by Western blot. It was observed that S. aureus-infected macrophages showed elevated ROS production, secretion of TNF-α, IL-1β, and CXCL8, along with increased expression of surface receptors (TNFR1, IL-1R, and CXCR1), and inflammatory markers (iNOS and COX-2) compared with control or treated groups (p

Aims to investigate α2-AR subtype distribution and the relationship between receptor amounts and their functionality in normotensive and spontaneously hypertensive rats. Methods experiments were performed on left ventricular cardiomyocytes isolated from Wistar rats and SHR (2–2.5 months). Molecular routine tools (RT-PCR, Western blotting, immunocytochemistry) were used for semi-quantitative estimation of α2-AR subtypes. Fluorescence of both the Ca2+-dependent and NO-sensitive probes were used to define functionality of α2-AR, evaluated by changes in the dynamics of spontaneous Ca2+-transients and NO production in cardiomyocytes in response to the α2-AR agonist application. Results percentage of the three known α2-AR subtypes in Wistar and SHR cardiomyocytes is not principally different. Total amounts of α2A-AR subtype in SHR increases, for both the sarcolemmal and intracellular receptor pools. Total number of α2B-AR is also significantly higher in hypertensive rats with an increase in the sarcolemmal, but not the intracellular immunoreactivity. For α2C-AR subtype, no significant differences between Wistar and SHR were identified, despite the fact that its amounts in cardiomyocytes are somewhat higher than the other two subtypes. Notwithstanding the increased expression of α2-AR subtypes in SHR, α2-AR-agonist guanabenz was ineffective in suppression of spontaneous Ca2+-transients, as well as the lowering of free calcium levels in the cytosol. Guanabenz-induced NO synthesis is well correlated with the Ca2+-loading into sarcoplasmic reticulum and actually decreased in SHR cardiomyocytes. Conclusion data indicate α2-AR dysfunction and ineffectiveness of α2-AR-mediated signaling pathways in this model of cardiovascular pathologies. Results can be used for clinical practice for more effective control of cardiovascular functions in various disease states.

The adhesion G protein-coupled receptor ADGRL1/latrophilin-1 (LPHN1) stabilizes synapse formation through heterophilic interactions. A growing consensus is pointing to the role of LPHN1 in modulating intracellular levels of cAMP, although conflicting data exist. Variants of LPHN1 resulting from alternative splicing differ at multiple sites, two of which, designated as SSA and SSB, modify extracellular and intracellular receptor regions, respectively. While SSA splicing modulates receptor-ligand affinity, the function of SSB splicing remains elusive. Here, we explored the role of SSB in an attempt to unify current findings on LPHN1 signaling pathways by testing SSB-containing and SSB-deficient receptor variants in signaling paradigms involving interaction with their ligands neurexin and FLRT. cAMP competitive binding assays revealed that cells expressing either receptor variant exhibited a ligand-dependent decrease in the forskolin-induced cAMP accumulation. Surprisingly, the expression of SSB-containing LPHN1 promoted both constitutive and ligand-dependent cAMP production, whereas SSB-deficient LPHN1 did not. Pertussis toxin treatment unveiled a constitutive coupling to Gαi/o for SSB-containing LPHN1 while abrogating the ligand-mediated activation of Gαs . Importantly, neither receptor variant increased the intracellular concentration of Ca2+ nor MAP kinase activation in the presence of ligands. These results suggest that SSB splicing selectively affects the duality of LPHN1 signaling toward opposing cAMP pathways.

Biochemistry, 58 (9), ISSN:0006-2960, ISSN:1520-4995

Background Breast cancer is the most common neoplasm in women and the most frequent cause of death in those between 35 and 55 years of age. All multicellular organisms have an innate immune system, whereas the adaptive or 'acquired' immune system is restricted to vertebrates. This study focused on the effect of conditioned medium isolated from cultured breast cancer cells on NB4 neutrophil-like cells. Materials and methods In the current study neutrophil-like NB4 cells were incubated with MCF-7 cell-conditioned medium. After 6 h incubation the intracellular receptor TLR2, was analyzed. Results The results revealed that MCF-7 cell-conditioned medium elicited expression of TLR2 in NB4 cells. Conclusions This treatment would result in the production of particular stimulants (i.e. soluble cytokines), eliciting the expression of immune system receptors. Furthermore, the flow cytometry results demonstrated that MCF-7 cell-conditioned medium elicited an effect on TLR2 intracellular receptors.

The aryl hydrocarbon receptor (AhR) was discovered as the intracellular receptor that bound with high affinity to the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the AhR is required for mediating the toxicity induced by TCDD. Subsequent studies show that the AhR binds structurally diverse chemicals including plant-derived compounds that promote health and several AhR-active pharmaceuticals that exhibit anticancer activity. In this issue, there is a report that carbidopa, a drug used for treating Parkinson's disease, is also an AhR ligand, and this compound inhibits pancreatic cancer cell and tumor growth. These results are consistent with activities of other AhR-active compounds that inhibit carcinogenesis. Like carbidopa, these chemicals are selective AhR modulators with potential clinical applications that are AhR-dependent.