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AMPA receptors (AMPARs) mediate the majority of excitatory transmission in the brain and enable the synaptic plasticity that underlies learning1. A diverse array of AMPAR signalling complexes are established by receptor auxiliary subunits, which associate with the AMPAR in various combinations to modulate trafficking, gating and synaptic strength2. However, their mechanisms of action are poorly understood. Here we determine cryo-electron microscopy structures of the heteromeric GluA1–GluA2 receptor assembled with both TARP-γ8 and CNIH2, the predominant AMPAR complex in the forebrain, in both resting and active states. Two TARP-γ8 and two CNIH2 subunits insert at distinct sites beneath the ligand-binding domains of the receptor, with site-specific lipids shaping each interaction and affecting the gating regulation of the AMPARs. Activation of the receptor leads to asymmetry between GluA1 and GluA2 along the ion conduction path and an outward expansion of the channel triggers counter-rotations of both auxiliary subunit pairs, promoting the active-state conformation. In addition, both TARP-γ8 and CNIH2 pivot towards the pore exit upon activation, extending their reach for cytoplasmic receptor elements. CNIH2 achieves this through its uniquely extended M2 helix, which has transformed this endoplasmic reticulum-export factor into a powerful AMPAR modulator that is capable of providing hippocampal pyramidal neurons with their integrative synaptic properties. Analyses of AMPA receptor–auxiliary subunit complexes provide insights into the gating and modulation of the AMPA receptor by TARP-γ8 and CNIH2.

The nanoparticle complex of cholesteryl pullulan (CHP) and NY-ESO-1 antigen protein (CHP-NY-ESO-1) presents multiple epitope peptides to MHC class I and II pathways, leading to CD8+ and CD4+ T cell responses. Poly-ICLC is a synthetic, double-stranded RNA, an agonist of toll-like receptor (TLR)-3, and a cytoplasmic receptor of melanoma differentiation-associated gene (MDA)-5. It should be a suitable immune adjuvant of cancer vaccine to overcome the inhibitory tumor microenvironment. We conducted a phase 1 clinical trial of CHP-NY-ESO-1 with poly-ICLC in patients with advanced or recurrent esophageal cancer. CHP-NY-ESO-1/poly-ICLC (μg/mg) was administered at a dose of 200/0.5 or 200/1.0 (cohorts 1 and 2, respectively) every 2 weeks for a total of six doses. The primary endpoints were safety and immune response. The secondary endpoint was tumor response. In total, 16 patients were enrolled, and six patients in each cohort completed the trial. The most common adverse event (AE) was injection site skin reaction (86.7%). No grade 3 or higher drug-related AEs were observed. No tumor responses were observed, and three patients (30%) had stable disease. The immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. Comparing CHP-NY-ESO-1 alone to the poly-ICLC combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. In a mouse model, adding anti-PD-1 antibody to the combination of CHP-NY-ESO-1/poly-ICLC suppressed the growth of NY-ESO-1-expressing tumors. Combining the vaccine with PD-1 blockade holds promise in human trials.

Plants have evolved numerous receptor-like kinases (RLKs) that modulate environmental stress responses. However, little is known regarding soybean (Glycine max) RLKs. We have previously identified that Glycine soja Ca2+ /CAM-binding RLK (GsCBRLK) is involved in salt tolerance. Here, we report that soluble NSF attachment protein receptor proteins BET1s mediate subcellular localization of calmodulin-binding receptor-like cytoplasmic kinases CRCK1s to modulate salt stress responses. Direct interaction between GsCBRLK and GsBET11a was initially identified via yeast two-hybrid and bimolecular fluorescence complementation assays. Further analysis demonstrated conserved interaction between BET1s and CRCK1s. GsCBRLK interacted with all BET1 proteins in wild soybean (Glycine soja) and Arabidopsis, and GsBET11a strongly associated with GsCRCK1a-1d, but slightly with AtCRCK1. In addition, GsBET11a interacted with GsCBRLK via its C-terminal transmembrane domain (TMD), where the entire TMD, not the sequence, was critical for the interaction. Moreover, the N-terminal variable domain (VD) of GsCBRLK was responsible for interacting with GsBET11a, and the intensity of interaction between GsCBRLK/AtCRCK1 and GsBET11a was dependent on VD. Furthermore, GsBET11a was able to mediate the GsCBRLK subcellular localization via direct interaction with VD. Additionally, knockout of AtBET11 or AtBET12 individually did not alter GsCBRLK localization, while GsBET11a expression caused partial internalization of GsCBRLK from the plasma membrane (PM). We further suggest the necessity of GsCBRLK VD for its PM localization via N-terminal truncation assays. Finally, GsBET11a was shown to confer enhanced salt stress tolerance when overexpressed in Arabidopsis and soybean. These results revealed the conserved and direct interaction between BET1s and CRCK1s, and suggested their involvement in salt stress responses.

Summary Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine‐rich repeat receptor kinases (LRR‐RK) FLS2 and EFR, and the LRR receptor protein (LRR‐RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families.We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR‐RK and LRR‐RP signaling.Quantitative differences in timing and amplitude were observed for several early immune responses, with RP‐mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23‐regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2‐signaling play similar roles in RLP23 signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP‐type immune receptors in a manner dependent on BIK1 kinase activity.Our study unveiled unexpected differences in two closely related receptor systems and reports a new negative role of BIK1 in plant immunity.

Protein kinase RNA-activated (PKR) is a cytoplasmic receptor for dsRNA, and as such is involved in detection of viral infection. On binding dsRNA, PKR dimerizes, autophosphorylates, and then phosphorylates its substrate, eukaryotic translation initiation factor 2 subunit α (eIF2α), causing inhibition of mRNA translation and shutdown of viral protein production. However, active PKR has also been found to be involved in the NF-κB signaling pathway by inducing phosphorylation of IκBα. PKR is regulated by the noncoding RNA nc886, which has altered expression in cancer. We have found that expression of nc886 is highly upregulated during activation of human CD4+ T cells. As has been described in other cell types, nc886 bound to PKR in human T cell lysates, preventing PKR phosphorylation by polyinosinic:polycytidylic acid or HIV trans-activation response element RNA in lysates of T cell lines or primary human CD4+ T cells. Using clonal human T cell lines, we found that nc886 expression was strictly required for IFN-γ and IL-2 expression and secretion after T cell activation but did not affect proliferation or activation-induced cell death. In stimulated human PBMCs, nc886 expression strongly correlated with IFN-γ expression. Although nc886 inhibited PKR activation by dsRNA, it was required for PKR phosphorylation during T cell stimulation, with subsequent NF-κB signaling and CREB phosphorylation. nc886 also regulated PKR phosphorylation during human monocyte-derived macrophage activation. We have therefore identified nc886 as a noncoding RNA marker of T cell activation and regulator of PKR-dependent signaling.

Prostate cancer is a major cause of death in males. Cyproterone acetate (CPA), the steroidal anti-androgen for part of androgen deprivation therapy, may block the androgen-receptor interaction and then reduce serum testosterone through its weak anti-gonadotropic action. In addition to CPA inducing hepatitis, CPA is known to cause liver tumors in rats also. Aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor and regulates multiple physiological functions. CYP1A1 is an AhR-targeted gene. We found that CPA induced CYP1A1 expression, transcriptional activity of the aryl hydrocarbon response element (AHRE), and the nuclear localization of AhR in mouse Hepa-1c1c7 cells. However, CPA suppressed CYP1A1 mRNA expression and the transcriptional activity of AHRE in human HepG2 and MCF7 cells, and also decreased AhR ligand-induced CYP1A1 protein expression and transcriptional activity of AHRE in HepG2 cells. In summary, CPA is an AhR agonist in mouse cells, but an AhR antagonist in human cells. Accordingly, CPA potentially plays a role as an endocrine disruptor of the AhR. This study helps us to understand why CPA induces acute hepatitis, gene mutation, and many other side effects. In addition, it may trigger further studies investigating the relationships between CPA, glucocorticoid receptor and castration-resistant prostate cancer in the future.

Legionella spp. are ubiquitous bacteria principally found in water networks and ∼20 species are implicated in Legionnaire’s disease. Among them, Legionella pneumophila is an intracellular pathogen of environmental protozoa, responsible for ∼90% of cases in the world. Legionella pneumophila regulates in part its virulence by a quorum sensing system named “Legionella quorum sensing,” composed of a signal synthase LqsA, two histidine kinase membrane receptors LqsS and LqsT and a cytoplasmic receptor LqsR. To date, this communication system was only found in L. pneumophila. Here, we investigated 58 Legionella genomes to determine the presence of a lqs cluster or homologous receptors using TBlastN. This analysis revealed three categories of species: 19 harbored a complete lqs cluster, 20 did not possess lqsA but maintained the receptor lqsR and/or lqsS, and 19 did not have any of the lqs genes. No correlation was observed between pathogenicity and the presence of a quorum sensing system. We determined by RT-qPCR that the lqsA gene was expressed at least in four strains among different species available in our laboratory. Furthermore, we showed that the lqs genomic region was conserved even in species possessing only the receptors of the quorum sensing system, indicating an ancestral acquisition and various loss dynamics during evolution. This system could therefore function in interspecific communication as well.

AMPA glutamate receptors (AMPARs) mediate the majority of excitatory transmission in the brain, and enable synaptic plasticity that underlies learning 1. A diverse array of AMPAR signaling complexes are established by receptor auxiliary subunits, associating in various combinations to modulate trafficking, gating and synaptic strength 2. However, their mechanisms of action are poorly understood. Here, we determine cryo-electron microscopy structures of the heteromeric GluA1/2 receptor assembled with both TARP-γ8 and CNIH2, the predominant AMPAR complex in the forebrain, in both resting and active states (at 3.2 and 3.7 Å, respectively). Consequential for gating regulation, two γ8 and two CNIH2 subunits lodge at distinct sites beneath the ligand-binding domains of the receptor tetramer, with site-specific lipids shaping each interaction. Activation leads to a stark asymmetry between GluA1 and GluA2 along the ion conduction path, and an outward expansion of the channel triggers counter-rotations of both auxiliary subunit pairs, that promotes the active-state conformation. In addition, both γ8 and CNIH2 pivot towards the pore exit on activation, extending their reach for cytoplasmic receptor elements. CNIH2 achieves this through its uniquely extended M2 helix, which has transformed this ER-export factor into a powerful positive AMPAR modulator, capable of providing hippocampal pyramidal neurons with their integrative synaptic properties.

Introduction Schizophrenia is recognized as one of the most important mental illnesses of the last century. Many genetic and environmental factors are involved in this condition. Recently, the genome-wide association study identified two significant genes LRP8 and CEP85L associated with psychiatric disorders. LRP8 (low-density lipoprotein receptor-related protein 8) acts as a cytoplasmic receptor for Reelin. Many studies have revealed that LRP8 was significantly related to schizophrenia and bipolar disorder in Chinese population. CEP85L standing for 'centrosomal protein 85 kDa-like' is another gene, which has been reportedly associated with BPD. Methods We performed a case-control study to analyze the association between rs5177 single-nucleotide polymorphism in the LRP8 gene plus the single-nucleotide polymorphism rs11756438 in the CEP85L gene and schizophrenia in the Iranian population. The genotype for rs5177 was determined by ARMS PCR method, while for rs11756438 genotype, it was determined by PCR-RFLP method after which statistical analysis was performed for each polymorphism. In rs5177, the CC genotype was susceptible to the disease while G allele was associated with disease protection. Results and Conclusion In rs11756438, the AA genotype was associated with disease susceptibility, while allele A did not have a significant association with the disease.

Receptor-like cytoplasmic kinases (RLCKs) are receptor kinases that lack extracellular ligand-binding domains and have emerged as a major class of signaling proteins that regulate plant cellular activities in response to biotic/abiotic stresses and endogenous extracellular signaling molecules. We have identified a rice RLCK (OsRLCK311) that was significantly higher in transgenic pSARK-IPT rice (Oryza sativa) that exhibited enhanced growth under saline conditions. Overexpression of OsRLCK311 full-length protein (RLCK311FL) and the C-terminus of OsRLCK311 (&Delta, N) in Arabidopsis confirmed its role in salinity tolerance, both in seedlings and mature plants. Protein interaction assays indicated that OsRLCK311 and &Delta, N interacted in-vivo with the plasma membrane AQP AtPIP2, 1. The RLCK311-PIP2, 1 binding led to alterations in the stomata response to ABA, which was characterized by more open stomata of transgenic plants. Moreover, OsRLCK311-&Delta, N effect in mediating enhanced plant growth under saline conditions was also observed in the perennial grass Brachypodium sylvaticum, confirming its role in both dicots and monocots species. Lastly, OsRLCK311 interacted with the rice OsPIP2, 1. We suggest that the rice OsRLCK311 play a role in regulating the plant growth response under saline conditions via the regulation of the stomata response to stress. This role seems to be independent of the RLCK311 kinase activity, since the overexpression of the RLCK311 C-terminus (&Delta, N), which lacks the kinase full domain, has a similar phenotype to RLCK311FL.

Funder: CSC Cambridge International Scholarship, Asymmetric astral microtubule organization drives the polarized orientation of the S. cerevisiae mitotic spindle and primes the invariant inheritance of the old spindle pole body (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome asymmetries featured in self-renewing stem cell divisions. We previously implicated Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most upstream determinant linking SPB age, functional asymmetry and fate. Here we used structured illumination microscopy and biochemical analysis to explore the asymmetric landscape of nucleation sites inherently built into the spindle pathway and under the control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the construction of the new SPB promotes the correct balance of nucleation sites between the nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently link correct SPB morphogenesis, age and fate.

All reported prostaglandin EP2 receptor antagonists have a purely orthosteric, competitive mode of action. Herein, we report the characterization of compound 1 (pubchem CID 664888) as the first EP2 antagonist that features a reversible, agonist dependent allosteric mode of action. Compound 1 displayed an unsurmountable inhibition of cAMP accumulation stimulated by different EP2 agonists in C6 glioma cells overexpressing human EP2 (C6G-hEP2). The degree of reduction of agonist potency and efficacy depended on the agonist employed. Negative allosteric modulation was not observed in C6G cells overexpressing human EP4, IP, or DP1 receptors. Moreover, in the murine microglial cell line that stably expresses human EP2 receptors (BV2-hEP2), compound 1 reduced the EP2 agonist-induced elevation of interleukin 6 (IL-6), IL-1β, and hEP2 mRNA levels and increased that of tumor necrosis factor (TNF)-α. Compound 1 was docked into a homology model of hEP2. The predicted binding site on the cytoplasmic receptor surface was similar to that of allosteric inhibitors of the β2-adrenergic, CC chemokine receptor 9 (CCR9), and CC chemokine receptor 2 (CCR2) receptors, which supports the notion of a conserved G-protein-coupled receptor (GPCR) binding pocket for allosteric inhibitors. As the first agonist dependent negative allosteric modulator of EP2 receptor, the structure of this compound may provide a basis for developing improved allosteric modulators of EP2 receptors.

Coronaviruses are a family of positive-sense RNA viruses with human and agricultural significance. Characterizing the mechanisms by which coronavirus infection dictates pathogenesis or counters the host immune response would provide targets for the development of therapeutics. Here, we show that the aryl hydrocarbon receptor (AhR) is activated in cells infected with a prototypic coronavirus, mouse hepatitis virus (MHV), resulting in the expression of several effector genes. AhR is important for modulation of the host immune response to MHV and plays a role in the expression of TiPARP, which we show is required for maximal viral replication. Taken together, our findings highlight a previously unidentified role for AhR in regulating coronavirus replication and the immune response to the virus., The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor/transcription factor that modulates several cellular and immunological processes following activation by pathogen-associated stimuli, though its role during virus infection is largely unknown. Here, we show that AhR is activated in cells infected with mouse hepatitis virus (MHV), a coronavirus (CoV), and contributes to the upregulation of downstream effector TCDD-inducible poly(ADP-ribose) polymerase (TiPARP) during infection. Knockdown of TiPARP reduced viral replication and increased interferon expression, suggesting that TiPARP functions in a proviral manner during MHV infection. We also show that MHV replication induced the expression of other genes known to be downstream of AhR in macrophages and dendritic cells and in livers of infected mice. Further, we found that chemically inhibiting or activating AhR reciprocally modulated the expression levels of cytokines induced by infection, specifically, interleukin 1β (IL-1β), IL-10, and tumor necrosis factor alpha (TNF-α), consistent with a role for AhR activation in the host response to MHV infection. Furthermore, while indoleamine 2,3-dioxygenase (IDO1) drives AhR activation in other settings, MHV infection induced equal expression of downstream genes in wild-type (WT) and IDO1−/− macrophages, suggesting an alternative pathway of AhR activation. In summary, we show that coronaviruses elicit AhR activation by an IDO1-independent pathway, contributing to upregulation of downstream effectors, including the proviral factor TiPARP, and to modulation of cytokine gene expression, and we identify a previously unappreciated role for AhR signaling in CoV pathogenesis. IMPORTANCE Coronaviruses are a family of positive-sense RNA viruses with human and agricultural significance. Characterizing the mechanisms by which coronavirus infection dictates pathogenesis or counters the host immune response would provide targets for the development of therapeutics. Here, we show that the aryl hydrocarbon receptor (AhR) is activated in cells infected with a prototypic coronavirus, mouse hepatitis virus (MHV), resulting in the expression of several effector genes. AhR is important for modulation of the host immune response to MHV and plays a role in the expression of TiPARP, which we show is required for maximal viral replication. Taken together, our findings highlight a previously unidentified role for AhR in regulating coronavirus replication and the immune response to the virus.

Enteric viruses including hepatitis E virus (HEV), human norovirus (HuNV), and rotavirus are causing global health issues. The host interferon (IFN) response constitutes the first-line defense against viral infections. Melanoma Differentiation-Associated protein 5 (MDA5) is an important cytoplasmic receptor sensing viral infection to trigger IFN production, and on the other hand it is also an IFN-stimulated gene (ISG). In this study, we investigated the effects and mode-of-action of MDA5 on the infection of enteric viruses. We found that MDA5 potently inhibited HEV, HuNV and rotavirus replication in multiple cell models. Overexpression of MDA5 induced transcription of important antiviral ISGs through IFN-like response, without triggering of functional IFN production. Interestingly, MDA5 activates the expression and phosphorylation of STAT1, which is a central component of the JAK-STAT cascade and a hallmark of antiviral IFN response. However, genetic silencing of STAT1 or pharmacological inhibition of the JAK-STAT cascade only partially attenuated the induction of ISG transcription and the antiviral function of MDA5. Thus, we have demonstrated that MDA5 effectively inhibits HEV, HuNV and rotavirus replication through provoking a non-canonical IFN-like response, which is partially dependent on JAK-STAT cascade.

The innate immune system plays a critical role in the early detection of pathogens, primarily by relying on pattern-recognition receptor (PRR) signaling molecules. Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic receptor that recognizes invading molecules and danger signals inside the cells. Recent studies highlight the importance of NOD2′s function in maintaining the homeostasis of human body microbiota and innate immune responses, including induction of proinflammatory cytokines, regulation of autophagy, modulation of endoplasmic reticulum (ER) stress, etc. In addition, there is extensive cross-talk between NOD2 and the Toll-like receptors that are so important in the induction and tuning of adaptive immunity. Polymorphisms of NOD2′s encoding gene are associated with several pathological conditions, highlighting NOD2′s functional importance. In this study, we summarize NOD2′s role in cellular signaling pathways and take a look at the possible consequences of common NOD2 polymorphisms on the structure and function of this receptor.

DAI-1 receptor (DNA-dependent activator of IFN-regulatory factors; DLM-1/ZBP-1) is an innate immunity cytoplasmic receptor of the DNA-recognition receptor class of antiviral immunity. DAI-1 expression reflects the severity of the inflammatory response that plays the key role in the pathogenesis of pregnancy complications. We studied DAI-1 receptor expression in the placental villi in early- and late-onset preeclampsia. In case of early-onset preeclampsia DAI-1 staining intensity was lower (p=0.01), and in case of late preeclampsia - significantly higher (p

Significance Viral-based delivery vectors have huge potential in the treatment of human disease. Adenoviral vectors specifically have proven highly efficacious in delivering corrected genes, as part of gene therapy, and vaccine epitopes for treating cancer and infectious disease. A principal obstacle to their widespread use is that antibodies potently neutralize them, limiting treatment to naïve patients. How antibodies block adenovirus-based transduction has long remained a mystery because, even though they prevent transgene expression, they do not prevent transgene delivery into target tissue. Here we show that the cytosolic antibody receptor TRIM21 is responsible for intercepting adenoviral gene therapy and vaccine vectors and neutralizing them. Gene KO of TRIM21 or a single-antibody mutation that prevents interaction is sufficient to restore transgene expression., Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is mediated by the cytosolic antibody receptor TRIM21. Genetic KO of TRIM21 or a single-antibody point mutation is sufficient to restore transgene expression to near-naïve immune levels. TRIM21 is also responsible for blocking cytotoxic T cell induction by vaccine vectors, preventing a protective response against subsequent influenza infection and an engrafted tumor. Furthermore, adenoviral preexisting immunity can lead to an augmented immune response upon i.v. administration of the vector. Transcriptomic analysis of vector-transduced tissue reveals that TRIM21 is responsible for the specific up-regulation of hundreds of immune genes, the majority of which are components of the intrinsic or innate response. Together, these data define a major mechanism underlying the preimmune block to adenovirus gene therapy and demonstrate that TRIM21 efficiently blocks gene delivery in vivo while simultaneously inducing a rapid program of immune transcription.

Neuropathic pain is one of the most important types of chronic pain. It is caused by neuronal damage. Clinical and experimental studies suggest a critical role for neuroimmune interactions in the development of neuropathic pain. In this article, we have shown that the cytoplasmic receptor Nod-like receptor-2, NOD2, and its adaptor-signaling molecule RIPK2 participate in the development of neuropathic pain after peripheral nerve injury (spared nerve injury model). The activation of NOD2 signaling in peripheral macrophage mediates the development of neuropathic pain through the production of pronociceptive cytokines (tumor necrosis factor and IL-1β). This study found that peripheral nerve injury promoted a systemic increase in the NOD2 ligand. These results highlight a previously undetermined role for NOD2 signaling in the development of neuropathic pain, suggesting a new potential target for preventing neuropathic pain.

Inflammasomes are positioned to rapidly escalate the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. However, negative regulation of inflammasomes remains poorly understood, as is the signaling cascade that dampens inflammasome activity. We found that rapid NLRP3 inflammasome activation was directly inhibited by protein kinase A (PKA), which was induced by prostaglandin E2 (PGE2) signaling via the PGE2 receptor E-prostanoid 4 (EP4). PKA directly phosphorylated the cytoplasmic receptor NLRP3 and attenuated its ATPase function. We found that Ser295 in human NLRP3 was critical for rapid inhibition and PKA phosphorylation. Mutations in NLRP3-encoding residues adjacent to Ser295 have been linked to the inflammatory disease CAPS (cryopyrin-associated periodic syndromes). NLRP3-S295A phenocopied the human CAPS mutants. These data suggest that negative regulation at Ser295 is critical for restraining the NLRP3 inflammasome and identify a molecular basis for CAPS-associated NLRP3 mutations.

Absent in melanoma 2 (AIM2) is a recently recognized cytoplasmic receptor which could sense cytoplasmic double-stranded DNA (dsDNA). After AIM2 detects the presence of parasitic nucleic acids (dsDNA) derived from invasive bacteria or viral genomes (for example, vaccinia virus and cytomegalovirus) within infected cells, AIM2 inflammasome could be formed. The formed AIM2 inflammasome could induce innate immune response and increase expressions of IL-1β and IL-18. Hepatitis B virus (HBV) is a hepatotropic, non-cytopathic double-stranded DNA virus. The immune response to viral antigens or virus is thought to be responsible for both liver damage and viral clearance in patients with HBV infection. However, there are no reports about whether AIM2 inflammasome exists in hepatocytes. In the present study, we investigated the presence and activity of AIM2 inflammasome in human hepatocytes. We found that AIM2 was expressed in cytoplasm of hepatocytes, and IL-18 expression was increased after AIM2 sensed HBV in hepatocytes in vitro. These results showed that AIM2 inflammasome was active in hepatocytes. We also found that hepatic AIM2 expression of chronic hepatitis B (CHB) patients was higher than that of controls. Hepatic AIM2 expression levels were positively correlated to the severity of liver inflammation. IL-18 is already considered to be associated with hepatic injury during HBV infection. In conclusion, we, therefore, believe that AIM2 inflammasome in hepatocytes might play an important role in the development and maintenance of HBV-related hepatitis.

Breast cancer (BC) patients experience increased stress with elevated cortisol levels, increasing risk of cancer recurrence. Cortisol binds to a cytoplasmic receptor, glucocorticoid receptor (GR) encoded by GR gene (NR3C1). We hypothesized that not only cancer cells, but even immune cells in the tumor microenvironment (TME) may contribute to GR expression in bulk tumor and influence prognosis. To test this, mRNA expression data was accessed from METABRIC and TCGA. &ldquo, High&rdquo, and &ldquo, low&rdquo, expression was based on highest and lowest quartiles of NR3C1 gene expression, respectively. Single-cell sequencing data were obtained from GSE75688 and GSE114725 cohorts. Computer algorithms CIBERSORT, Gene Set Enrichment Analysis and TIMER were used. GR-high BC has better median disease-free and disease-specific survival. Single cell sequencing data showed higher GR expression on immune cells compared to cancer and stromal cells. Positive correlation between GR-high BC and CD8+ T-cells was noted. In GR-high tumors, higher cytolytic activity (CYT) with decreased T-regulatory and T-follicular helper cells was observed. High GR expression was associated with lower proliferation index Ki67, enriched in IL-2_STAT5, apoptosis, KRAS, TGF-&beta, signaling, and epithelial-to-mesenchymal transition. Immune cells significantly contribute to GR expression of bulk BC. GR-high BC has a favorable TME with higher CYT with favorable outcomes.

Most organisms on Earth use glucose, a photosynthetic product, as energy source. The chloroplast, the home of photosynthesis, is the most representative and characteristic organelle in plants and is enclosed by the outer envelope and inner envelope membranes. The chloroplast biogenesis and unique functions are very closely associated with proteins in the two envelope membranes of the chloroplast. Especially, the chloroplast outer envelope membrane proteins have important roles in signal transduction, protein import, lipid biosynthesis and remodeling, exchange of ions and numerous metabolites, plastid division, movement, and host defense. Therefore, biogenesis of these membrane proteins of chloroplast outer envelope membrane is very important for biogenesis of the entire chloroplast proteome as well as plant development. Most proteins among the outer envelope membrane proteins are encoded by the nuclear genome and are post-translationally targeted to the chloroplast outer envelope membrane. In this process, cytoplasmic receptor and import machineries are required for efficient and correct targeting of these membrane proteins. In this review, we have summarized recent advances on the sorting, targeting, and insertion mechanisms of the outer envelope membrane proteins of chloroplasts and also provide future direction of the study on these topics.

Macrophages are versatile phagocytic cells in immune system with immunoregulatory functions. However, the removal of apoptotic cells by macrophages is disturbed in systemic lupus erythematosus (SLE). Aryl hydrocarbon receptor (AhR) is a ligand-activated cytoplasmic receptor and transcription factor with diverse effects on immune response. Indole-3-carbinol (I3C) is an AhR agonist which has been implicated as a beneficial factor in regulating inflammation and cytokine expression in murine models of SLE. However, the molecular mechanisms are not thoroughly studied. Here, we aimed to investigate the ex vivo effects of I3C on polarization of monocyte-derived macrophages (MDMs) in SLE patients and the expression of regulatory cytokines upon AhR activation. MDMs from 15 newly diagnosed SLE patients and 10 normal subjects were induced by Jurkat apoptotic bodies (JABs) and treated with I3C. I3C enhanced the nuclear accumulation of AhR among MDMs of SLE patients and altered the expression of AhR target genes including CYP1A1, IL1- β, IDO-1 and MRC-1. The imbalanced expression of pro- and anti- inflammatory cytokines (IL-10, IL-12, TGFβ1, TNFα, IL-23, IL-6 and IFN-γ) was compensated in response to I3C. AhR activation was also associated with the overexpression of M2 markers (CD163) and downregulation of M1 markers (CD86). Thus, macrophages are activated alternatively in response to I3C. The obtained data indicate that I3C-mediated AhR activation possess immunoregulatory effects on macrophages of SLE patients by exerting an obvious downregulation in the expression of pro-inflammatory and overexpression of anti-inflammatory cytokines. Therefore, AhR could be targeted and further investigated as a choice of anti-inflammatory therapies for autoimmune disorders such as SLE.

The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor and transcription factor, primarily activated through cognate ligand binding. It is an important factor in immunity and tissue homeostasis, and structurally diverse compounds from the environment, diet, microbiome, and host metabolism, can influence AhR activity. Emerging evidence suggests that AhR is a key sensor allowing immune cells to adapt to environmental conditions. Changes in AhR activity have been associated with autoimmune disorders and cancer, and AhR agonists or antagonists impacting immune disease outcomes have placed AhR as a potential actionable target for immunotherapy. We describe known ligands stimulating AhR activity, downstream pro-inflammatory and suppressive mechanisms potentiated by AhR, and how this understanding is applied to immunopathologies. Indeed, AhR-dependent immune responses might be modulated to help control pathologic outcomes.

The causative agent of Chagas' disease, Trypanosoma cruzi, affects approximately 10 million people living mainly in Latin America, with macrophages being one of the first cellular actors confronting the invasion during T. cruzi infection and their function depending on their proper activation and polarization into distinct M1 and M2 subtypes. Macrophage polarization is thought to be regulated not only by cytokines and growth factors but also by environmental signals. The metabolic checkpoint kinase mammalian target of rapamycin (mTOR)-mediated sensing of environmental and metabolic cues influences macrophage polarization in a complex and as of yet incompletely understood manner. Here, we studied the role of the mTOR pathway in macrophages during T. cruzi infection. We demonstrated that the parasite activated mTOR, which was beneficial for its replication since inhibition of mTOR in macrophages by different inhibitors decreased parasite replication. Moreover, in rapamycin pretreated and infected macrophages, we observed a decreased arginase activity and expression, reduced IL-10 and increased interleukin-12 production, compared to control infected macrophages treated with DMSO. Surprisingly, we also found a reduced iNOS activity and expression in these macrophages. Therefore, we investigated possible alternative mechanisms involved in controlling parasite replication in rapamycin pretreated and infected macrophages. Although, cytoplasmic ROS and the enzyme indoleamine 2, 3-dioxygenase (IDO) were not involved, we observed a significant increase in IL-6, TNF-α, and IL-1β production. Taking into account that IL-1β is produced by activation of the cytoplasmic receptor NLRP3, which is one of the main components of the inflammasome, we evaluated NLRP3 expression during mTOR inhibition and T. cruzi infection. We observed that rapamycin-pretreated and infected macrophages showed a significant increase in NLRP3 expression and produced higher levels of mitochondrial ROS (mtROS) compared with control cells. Moreover, inhibition of mtROS production partially reversed the effect of rapamycin on parasite replication, with there being a significant increase in parasite load in rapamycin pretreated and infected macrophages from NLRP3 KO mice compared to wild-type control cells. Our findings strongly suggest that mTOR inhibition during T. cruzi infection induces NLRP3 inflammasome activation and mtROS production, resulting in an inflammatory-like macrophage profile that controls T. cruzi replication. Fil: Rojas Marquez, Jorge David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Ana, Yamile. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Baigorri, Ruth Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Stempin, Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Cerban, Fabio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

International audience; Inflammasomes are intracellular multiprotein signaling complexes, mainly present in myeloid cells. They commonly assemble around a cytoplasmic receptor of the nucleotide-binding leucine-rich repeat containing receptor (NLR) family, although other cytoplasmic receptors like pyrin have been shown to form inflammasomes. The nucleation of the multiprotein scaffolding platform occurs upon detection of a microbial, a danger or a homeostasis pattern by the receptor that will, most commonly, associate with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) through homotypic domain interactions resulting in recruitment of procaspase-1. This will lead to the autoproteolytic activation of caspase-1, which regulates the secretion of proinflammatory IL1β and IL18 cytokines and pyroptosis, a caspase-1-mediated form of cell death. Pyroptosis occurs through cleavage of Gasdermin D, a membrane pore forming protein. Recently, non-canonical inflammasomes have been described, which directly sense intracellular pathogens through caspase-4 and -5 in humans, leading to pyroptosis. Inflammasomes are important in host defense; however, a deregulated activity is associated with a number of inflammatory, immune and metabolic disorders. Furthermore, mutations in inflammasome receptor coding genes are causal for an increasing number of rare autoinflammatory diseases. Biotherapies targeting the products of inflammasome activation as well as molecules that directly or indirectly inhibit inflammasome nucleation and activation are promising therapeutic areas. This review discusses recent advances in inflammasome biology, the molecular pathology of several inflammasomes, and current therapeutic approaches in autoinflammatory diseases and in selected common multifactorial inflammasome-mediated disorders.

International audience; The aryl hydrocarbon receptor (or AhR) is a cytoplasmic receptor of pollutants. It translocates into the nucleus upon binding to its ligands, and forms a heterodimer with ARNT (AhR nuclear translocator). The heterodimer is a transcription factor, which regulates the transcription of xenobiotic metabolizing enzymes. Expressed in many cells in vertebrates, it is mostly present in neuronal cell types in invertebrates, where it regulates dendritic morphology or feeding behavior. Surprisingly, few investigations have been conducted to unravel the function of the AhR in the central or peripheral nervous systems of vertebrates. In this review, we will present how the AhR regulates neural functions in both invertebrates and vertebrates as deduced mainly from the effects of xenobiotics. We will introduce some of the molecular mechanisms triggered by the well-known AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which impact on neuronal proliferation, differentiation, and survival. Finally, we will point out the common features found in mice that are exposed to pollutants, and in AhR knockout mice.

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.

Neutrophils use a broad array of pattern recognition receptors to sense and respond to invading pathogens and are important in the early control of acute bacterial infections. Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan. Reduced neutrophil NOD1 expression has been reported in periparturient dairy cows. The aim of this study was to investigate the role of NOD1 signalling in the early responses of bovine neutrophils to bacterial infections. Blood neutrophils from healthy heifers were preincubated for 2h with ML130, a selective inhibitor of NOD1-dependent nuclear factor-κB (NF-κB) activation. Thereafter, cells were cultured with live Escherichia coli for additional 30 min or subjected to Boyden chamber cell migration assay with E. coli in the lower chamber. Results showed that ML130 inhibited E. coli-induced NF-κB nuclear translocation. There was an indication, although not significant, that ML130 down-regulated gene expression of proinflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, chemokines IL-8 and C-X-C motif ligand 2 (CXCL2), and adhesion molecule CD62L, in E. coli-challenged neutrophils. Flow cytometry-based Annexin V staining revealed a considerable increase in neutrophil survival upon E. coli infection, an effect that was attenuated in the presence of ML130. Additionally, inhibition of NOD1/NF-κB signalling resulted in reduced migration of neutrophils to E. coli, and impaired phagocytosis, intracellular bacterial killing and reactive oxygen species production by neutrophils. These results indicate that NOD1/NF-κB pathway plays a crucial role in modulating neutrophil responses that are important for early control of infections. Approaches aiming at restoring neutrophil NOD1 function could be beneficial for prevention or treatment of coliform mastitis.

Objective Nucleotide-binding oligomerization domain (NOD) protein, as cytoplasmic receptor of the innate immune response, plays an important role in adipose inflammation and insulin resistance in obesity. Our objective was to examine adipose tissue (AT) NOD in nascent metabolic syndrome (MetS) patients and to investigate its association with MetS features. Methods Thirty-four MetS subjects and 31 controls were recruited. Fasting blood was collected, and abdominal subcutaneous AT was obtained by biopsy for NOD1/NOD2 expression and activity. Results MetS subjects showed significantly increased expression for NOD1 on adipose depots as compared to controls. In addition to increased expression of downstream signaling mediators RIPK2 and NF-κB p65 nuclear translocation, there was remarkably higher release of monocyte chemotactic protein1 (MCP-1), interleukin (IL)-6, and IL-8 in MetS versus controls following priming of the isolated adipocytes with NOD1 ligand iE-DAP. With regard to NOD2, the differences between the two groups were not significant in either basal state or after activation. Increased NOD1 positively correlated with waist circumference. NOD1 was also correlated with HbA1c and HOMA-IR. NOD1 positively correlated with serum levels of IL-6, MCP-1, and NF-κB activity. Conclusions Activation of the innate immune pathway via NOD1 may be partially responsible for the increased systemic inflammation and insulin resistance in MetS.

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on "conformationally constrained" GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function.

Keratinocytes are routinely subjected to both internal and external stimulation. This study investigates the effects of interferon gamma, interleukin-4, tumor necrosis factor alpha, and the synthetic immunomodulator muramyl dipeptide on the human keratinocyte cell line, HaCaT. Following HaCaT stimulation with cytokines or muramyl dipeptide for different time periods, changes in the expression of different cell surface receptors, cell proliferation, and cell apoptosis were evaluated by flow cytometry, tritiated thymidine uptake, and annexin-V staining, respectively. A significant decrease in the expression of CD49d was found upon treatment with interleukin-4. Interferon gamma and tumor necrosis factor alpha increased the expression of intercellular adhesion molecule 1 and major histocompatibility complex class I, whereas major histocompatibility complex class II and CD1b were only upregulated by interferon gamma. Interferon gamma and tumor necrosis factor alpha had opposite effects regarding CD119 expression, with the former downregulating, while the latter upregulating its expression. Of the stimuli tested, only interferon gamma and tumor necrosis factor alpha significantly inhibited proliferation of HaCaT cells, yet only interferon gamma played a significant role in inducing HaCaT cell apoptosis. Our data demonstrate differential effects of the three tested cytokines on keratinocytes and reveal that the absence of HaCaT cell responses to muramyl dipeptide is associated with undetectable levels of its cytoplasmic receptor, nucleotide-binding oligomerization domain–containing protein 2.

Background: The pharmacology activity of corticosteroids, is due to the formation in the blood of the complex Corticosteroid- Protein Glycosylated, that, in this form, after the binding to the cytoplasmic receptor, penetrates in the target cells. This interaction process, also happens, with salivary proteins The aim of this study is, to study this process, that precipitate the salivary proteins, and with them, the salivary secreted mucin. Materials and methods: In two samples of whole saliva provided by volunteers, are added different concentrations of three corticosteroids, beclometasone, budesonide, fluticasone. The samples are centrifuged, and in surnatant, dosed, the amounts of total salivary proteins and mucins. The results are statistically analyzed with Mann Whitney U Test, Test T, Pearson Correlation Coefficient Results and discussion: With all dosage, the difference of the proteins and mucins precipitated by the Budesonide, and beclometasone vs fluticasone, are statistically different., p ≤ 0.05. For all three corticosteroids, there is a saturation value, with a good correlation between corticosteroids’s dosage and the amount of the proteinmucins precipitation, (Pearson coefficient of 0.91). The little difference in the precipitation of the mucins, and the proteins, p=0.0334, obtained with the budesonide versus beclometasone, can find an explanation, for the presence in the first, of two hydroxyl groups, (one in beclometasone). The difference of beclometasone and budesonide, versus fluticasone, is due assuming that the parameters, that stabilize the (CCP), type hydrogen bonds and Van der Waals forces, are more influenced by solubility in water, there is nothing for the fluticasone, rather than by the chemical conformation of drugs

Immunity to microbial infection is a common feature of metazoans and plants. Plants employ plasma membrane and cytoplasmic receptor systems for sensing microbe-derived or host-derived patterns and effectors and to trigger inducible immune defenses. Different biochemical types of plasma membrane immune receptors mediate recognition predominantly of peptide and carbohydrate patterns. Current research highlights the role of immune receptor complex formation in plant immunity. In particular, ligand binding by immune receptors generates molecular surfaces that enable either receptor homo-dimerization or co-receptor recruitment for subsequent signal transduction. New insight into negative regulatory principles of immune receptor function further suggests substantial dynamics in protein-protein interactions at the plasma membrane that we are only beginning to understand.

Constitutive activation of the Jak/Stat signalling pathway is a hallmark of Myeloproliferative Neoplasms (MPN), with 60-90% of patients carrying the activating JAK2 mutation, JAK2V617F. Several studies suggest expression of a type I cytokine receptor is essential for JAK2V617F-mediated transformation, but the precise contribution of such receptors is unresolved. Here we investigate the functional role of Mpl in JAK2V617F-driven MPN in vitro and in vivo. In vitro studies confirmed that Mpl expression was required for JAK2V617F-induced transformation, but identified a Mpl truncation mutant retaining the JAK2-interaction domain, but lacking the C-terminus (Mpl-IC36), typically associated with Stat5 binding, to be sufficient to support JAK2V617F-induced transformation. Interestingly, Stat5-phosphorylation was maintained. In contrast, deletion of the entire cytoplasmic receptor domain abrogated Stat5 phosphorylation and factor-independent proliferation. In vivo Mpl-wt;JAK2V617F mice exhibited the thrombocytosis and erythrocytosis typical of MPN, while Mpl deficiency abrogated thrombocytosis but sustained high red blood cell counts. Although the Mpl-IC36 receptor was sufficient for JAK2V617F-induced factor independence in BaF/3 cells, Mpl-IC36;JAK2V617F mice did not develop thrombocytosis, indeed platelet counts were similar to those in Mpl null mice. This underlines differences that may emerge from in vitro and in vivo systems and highlights the important role of the Mpl cytoplasmic region for JAK2V617F-mediated signalling, extending the function of Mpl in JAK2V617F-induced MPN beyond a simple scaffold for active JAK2.

Melanoma differentiation associated gene 5 (MDA5) is an important cytoplasmic receptor that recognizes long molecules of viral double-stranded RNA and single-stranded RNA with 5′ triphosphate and mediates type I interferon secretion. In this study, the full-length MDA5 gene in the goose was identified and characterized. The cDNA of goose MDA5 was 3,306 bp in length with an open reading frame of 3,018 bp, which encoded a polypeptide of 1,005 amino acids. The deduced amino acid sequence contained 6 main structure domains including 2 caspase activation and recruitment domains, one DExD/H-box helicase domain, one type III restriction enzyme domain, one helicase conserved C-terminal domain, and one RIG-I C-terminal domain. Quantitative real-time PCR analysis indicated that goose MDA5 mRNA was constitutively expressed in all sampled tissues. It was highly expressed in the jejunum, trachea, ileum, colon, and kidney, and lowly expressed in the muscular stomach, glandular stomach, and muscle. A significant increase in the transcription of MDA5 was detected in the brain, spleen, and lungs of geese after infection with H5N1 highly pathogenic avian influenza virus compared with uninfected tissues. These findings indicated that goose MDA5 was an important receptor, involved in the antiviral innate immune defense to H5N1 highly pathogenic avian influenza virus in geese.

The wall-associated kinases (WAKs)1 are receptor protein kinases that bind to long polymers of cross-linked pectin in the cell wall. These plasma-membrane-associated protein kinases also bind soluble pectin fragments called oligo-galacturonides (OGs) released from the wall after pathogen attack and damage. WAKs are required for cell expansion during development but bind water soluble OGs generated from walls with a higher affinity than the wall-associated polysaccharides. OGs activate a WAK-dependent, distinct stress-like response pathway to help plants resist pathogen attack. In this report, a quantitative mass-spectrometric-based phosphoproteomic analysis was used to identify Arabidopsis cellular events rapidly induced by OGs in planta. Using N14/N15 isotopic in vivo metabolic labeling, we screened 1,000 phosphoproteins for rapid OG-induced changes and found 50 proteins with increased phosphorylation, while there were none that decreased significantly. Seven of the phosphosites within these proteins overlap with those altered by another signaling molecule plants use to indicate the presence of pathogens (the bacterial “elicitor” peptide Flg22), indicating distinct but overlapping pathways activated by these two types of chemicals. Genetic analysis of genes encoding 10 OG-specific and two Flg22/OG-induced phosphoproteins reveals that null mutations in eight proteins compromise the OG response. These phosphorylated proteins with genetic evidence supporting their role in the OG response include two cytoplasmic kinases, two membrane-associated scaffold proteins, a phospholipase C, a CDPK, an unknown cadmium response protein, and a motor protein. Null mutants in two proteins, the putative scaffold protein REM1.3, and a cytoplasmic receptor like kinase ROG2, enhance and suppress, respectively, a dominant WAK allele. Altogether, the results of these chemical and genetic experiments reveal the identity of several phosphorylated proteins involved in the kinase/phosphatase-mediated signaling pathway initiated by cell wall changes.