Component Of Receptor Complex Research Articles

Messenger RNA transcription levels of neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex components in the olfactory nerve system of the anadromous Pacific salmon, masu salmon Oncorhynchus masou.

Anadromous Pacific salmon (genus Oncorhynchus) are known for homing behavior to their natal rivers based on olfactory imprinted memories during seaward migration. The SNARE complex is a regulator of vesicle exocytosis from the presynaptic membrane. Our previous study suggested that its component genes (Snap25, Stx1, and Vamp2) are more highly expressed in the olfactory nervous system (ONS) during the migration stages associated with olfactory imprinting in the evolutionary species of Pacific salmon, such as chum (O. keta) and pink (O. gorbuscha) salmon. Masu salmon (O. masou) has a significantly different life history from these species, living longer in rivers and being a more primitive Pacific salmon species. In this study, the transcription of snare mRNAs in the ONS was analyzed using mainly male wild masu salmon. Five cDNAs encoding masu salmon SNAREs, which are well conserved among vertebrates, were isolated and sequenced. Each snare mRNA was highly expressed in age 1+ (yearling) parr prior to smoltification, particularly in the olfactory bulb. Their transcription status was significantly different from that of chum and pink salmon, which showed high expression in earlier under-yearling juveniles. The present results and our previous studies indicate that snare mRNAs are highly transcripted until the seaward migration, reflecting neural development and neuroplasticity of the ONS for olfactory imprinting.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.

Macrophage inflammarafts in atherosclerosis.

Advances in single cell techniques revealed a remarkable diversity in macrophage gene expression profiles in atherosclerosis. However, the diversity of functional processes at the macrophage plasma membrane remains less studied. This review summarizes recent advances in characterization of lipid rafts, where inflammatory receptors assemble, in macrophages that undergo reprogramming in atherosclerotic lesions and in vitro under conditions relevant to the development of atherosclerosis. The term inflammarafts refers to enlarged lipid rafts with increased cholesterol content, hosting components of inflammatory receptor complexes assembled in close proximity, including TLR4-TLR4, TLR2-TLR1 and TLR2-CD36 dimers. Macrophages decorated with inflammarafts maintain chronic inflammatory gene expression and are primed to an augmented response to additional inflammatory stimuli. In mouse atherosclerotic lesions, inflammarafts are expressed primarily in nonfoamy macrophages and less in lipid-laden foam cells. This agrees with the reported suppression of inflammatory programs in foam cells. In contrast, nonfoamy macrophages expressing inflammarafts are the major inflammatory population in atherosclerotic lesions. Discussed are emerging reports that help understand formation and persistence of inflammarafts and the potential of inflammarafts as a novel therapeutic target. Chronic maintenance of inflammarafts in nonfoamy macrophages serves as an effector mechanism of inflammatory macrophage reprogramming in atherosclerosis.

PS-B12-11: FUNCTIONAL VERSATILITY OF (PRO) RENIN RECEPTOR CLARIFIED BY STRUCTURAL AND EVOLUTIONARY ANALYSES

Objective: (Pro)renin receptor [(P)RR] is a single-span membrane protein originally identified as a regulator of the renin-angiotensin-aldosterone system. (P)RR reportedly exerts various functions, including an accessory protein of vacuolar ATPase complex and a component of Wnt receptor complex. The soluble form of (P)RR [s(P)RR] which comprises of most of extracellular domain (ECD) exerts an antidiuretic action. although the versatile functions should be mediated through the interactions between (p)rr ecd and its respective binding partners, the one-to-many bindings has not been fully explained. Here we investigated the binding mode of this receptor by structural and evolutionary analyses using three-dimensional structural models of (P)RR, which were predicted by the AlphaFold2 (AF2) program, a machine learning-based algorithm capable of predicting protein structure to near experimental accuracy. Design and method: Monoclonal antibodies against (P)RR reduce the Wnt/beta-catenin-dependent development of pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic cancer. Antibodies against two (P)RR regions (residues 47–60 and 200–213) located in ECD reduce the proliferation of human PDAC cells in vitro. The spatial distribution of the two regions was examined using human (P)RR AF2 structural models (monomer and dimer). The evolutionary conservation profile and hydrophobicity were mapped on the models. Results: The dimer structure predicted by AF2 shows that (P)RR homodimerizes via ECD, which explains the experimentally proven dimer formation. The 47–60 and 200–213 regions of (P)RR formed a continuous surface patch on the structural model, which comprised evolutionarily conserved hydrophobic residues. The dimer model revealed the presence of two hand-like grooves, with 47–60 and 200–213 regions as its palm and 270–296 region (intrinsically disordered region, IDR) as its fingers. Conclusions: Considering conformational flexibility of IDR allowing for multiple protein interactions, we propose that the grooves may act as a binding interface with Wnt signaling molecules and antibodies against the grooves probably interfere with the formation of Wnt receptor complex due to steric hindrance and direct competition with the binding interface residues. The hydrophobic nature of the grooves may explain why (P)RR shows one-to-many bindings underpinning its functional versatility. This study provides new insights into the binding mode of (P)RR for the first time. s(P)RR is considered a useful biomarker for diseases, including essential hypertension and cancer. Our findings may help explore a new treatment and biomarker testing of human diseases, and contribute to quality of life in a longevity society.

The oral secretion from Cotton Boll Weevil (Anthonomus grandis) induces defense responses in cotton (Gossypium spp) and Arabidopsis thaliana

Cotton Boll Weevil (CBW, Anthonomus grandis) is a devastating insect-pest affecting cotton (Gossypium spp), using cotton internal floral structures for larval development, making it difficult to control. Little is known about the mechanism underlying CBW-plant interactions leading to infestation or resistance. Here, we investigated the plant molecular responses triggered by CBW extracts from eggs, larvae, and oral secretions (OS) of larvae and adult. The MAPK pathway, one of the early signaling events in plant pattern-triggered immunity (PTI), was activated by CBW extracts in Arabidopsis thaliana. In cotton, CBW OS activated GhMPK3 and GhMPK6. Importantly, CBW OS activated the PTI marker gene FRK1 and the effector-triggered immunity (ETI) marker gene WRKY46 in Arabidopsis reporter lines, suggesting that Herbivore-Associated Molecular Patterns (HAMPs) present in OS from CBW can elicit plant defense responses in cotton and Arabidopsis. Interestingly, CBW OS-induced MAPK activation shows similar patterns in wild-type and Arabidopsis mutants, which bear mutations in already known receptor and co-receptors complex components, suggesting a distinct signaling cascade likely triggered by the putative HAMPs present in CBW OS. The work points to potentials in the identification and development of CBW extract-derived elicitors for effective pest control combating CBW infestation.

Klotho in Osx+-mesenchymal progenitors exerts pro-osteogenic and anti-inflammatory effects during mandibular alveolar bone formation and repair

Maxillofacial bone defects are commonly seen in clinical practice. A clearer understanding of the regulatory network directing maxillofacial bone formation will promote the development of novel therapeutic approaches for bone regeneration. The fibroblast growth factor (FGF) signalling pathway is critical for the development of maxillofacial bone. Klotho, a type I transmembrane protein, is an important components of FGF receptor complexes. Recent studies have reported the presence of Klotho expression in bone. However, the role of Klotho in cranioskeletal development and repair remains unknown. Here, we use a genetic strategy to report that deletion of Klotho in Osx-positive mesenchymal progenitors leads to a significant reduction in osteogenesis under physiological and pathological conditions. Klotho-deficient mensenchymal progenitors also suppress osteoclastogenesis in vitro and in vivo. Under conditions of inflammation and trauma-induced bone loss, we find that Klotho exerts an inhibitory function on inflammation-induced TNFR signaling by attenuating Rankl expression. More importantly, we show for the first time that Klotho is present in human alveolar bone, with a distinct expression pattern under both normal and pathological conditions. In summary, our results identify the mechanism whereby Klotho expressed in Osx+-mensenchymal progenitors controls osteoblast differentiation and osteoclastogenesis during mandibular alveolar bone formation and repair. Klotho-mediated signaling is an important component of alveolar bone remodeling and regeneration. It may also be a target for future therapeutics.

Alpha-Klotho protein in systemic lupus erythematosus.

Alpha-Klotho protein (α-Klotho) is an essential component of endocrine fibroblast growth factor receptor complexes that governs multiple metabolic processes including aging-related disorders, diabetes, cancer, arteriosclerosis, and chronic kidney disease. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can affect almost any organ in the body and in which multiple pathophysiological abnormalities are observed. In the present work, our objective was to study whether the serum levels of α-Klotho differ between patients with SLE and controls, and how this protein is related to the clinical and laboratory characteristics of the disease. Cross-sectional study that included 364 women, 195 of them diagnosed with SLE and 169 sex- and age-matched controls. Circulating α-Klotho was analysed in SLE patients and controls. A multivariable analysis was performed to assess whether α-Klotho differs between patients and controls, and to study its relationship with SLE features. No differences were found in α-Klotho levels between SLE patients and controls, both in univariable and multivariable analyses. Disease-related data like SLE duration, acute phase reactants, activity, severity and damage indices, and autoantibodies profile were not significantly associated with serum levels of α-Klotho. However, the use of prednisone and the presence of musculoskeletal manifestations were significantly related to higher α-Klotho serum levels. α-Klotho protein serum levels do not differ between patients with SLE and controls. Nevertheless, SLE patients taking prednisone or those with musculoskeletal manifestations show significantly higher circulating levels of α-Klotho.

MYC Hyperactivates Wnt Signaling in APC/CTNNB1-Mutated Colorectal Cancer Cells through miR-92a-Dependent Repression of DKK3.

Activation of Wnt signaling is among the earliest events in colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding β-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations affecting APC, a major β-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct β-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that MYC suppresses DKK3 transcription through one of miR-17-92 cluster miRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in Apc-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration/invasion in vitro and metastasis in a syngeneic orthotopic mouse colon cancer model. IMPLICATIONS: Our results suggest that the MYC→miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feed-forward oncogenic loop.

EXTH-10. EXPLORATION OF A NOVEL TOXIN-INCORPORATING CAR T CELL: HOW DOES CHLOROTOXIN RECOGNIZE GLIOBLASTOMA CELLS?

Abstract Chlorotoxin, a peptide toxin component of scorpion venom, binds selectively to glioblastoma and other neuroepithelial tumors, with minimal binding to non-malignant cells. We have recently developed a CAR T cell incorporating chlorotoxin (CLTX) as its target recognition domain, and CLTX-CAR T cells are now in clinical trial for recurrent glioblastoma (NCT04214392). We determined in preclinical studies that surface MMP-2 was required for CLTX-CAR T cell killing. However, the precise composition and structure of the cell surface complex recognized by CLTX-CAR T cells remains an unresolved question of increasing importance. Previous investigations have variously proposed matrix metalloprotease-2 (MMP-2), ClC-3 chloride channels, regulators of MMP-2, annexin A2, and neuropilin-1, as receptors, or components of receptors, for CLTX. To approach this question, we have visualized bound Cy5.5-conjugated CLTX peptide (CLTX.Cy5.5) or biotin-conjugated CLTX peptide (CLTX.biotin) on tumor cells in fixed sections of patient resections, on tumor cells in organotypic cultures of patient resections, and on cells of cultured patient-derived glioblastoma tumor lines. At tissue- and cell-level spatial resolution, we saw good correlation of CLTX binding with MMP-2 expression in patient tumor samples and on cultured GBM cells, and between CLTX binding and tumor cell death (cleaved caspase-3) in organotypic GBM cultures. However, at subcellular resolution, surface binding of CLTX was related to, but not precisely overlapping, with MMP-2 or other putative receptors. Rather, on fixed PBT003, PBT030 and PBT106 cells in monolayer cultures, MMP-2 staining was clustered, with diffuse loosely associated CLTX.biotin staining. Diffuse distribution of CLTX.Cy5.5 was also seen in fixed xenograft sections of PBT003-4, PBT1206, PBT030-2, PBT051 and PBT138 tumor cells, and not obviously associated with more discrete staining for IL13Rα2 and EGFR. Ongoing experiments are further examining association of CLTX with other putative components of CLTX receptor complexes, and their redistribution during binding by CLTX and CLTX-CAR T cells.

RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy.

RNF43 is an E3 ubiquitin ligase and known negative regulator of WNT/β-catenin signaling. We demonstrate that RNF43 is also a regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1, and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor and inhibits ROR2 activation. These activities of RNF43 are physiologically relevant and block pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E and MEK inhibitors. Next, RNF43 acted as melanoma suppressor and improved response to targeted therapies in vivo. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have a worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.

Phosphorylation-dependent routing of RLP44 towards brassinosteroid or phytosulfokine signalling.

ABSTRACTPlants rely on cell surface receptors to integrate developmental and environmental cues into behaviour adapted to the conditions. The largest group of these receptors, leucine-rich repeat receptor-like kinases, form a complex interaction network that is modulated and extended by receptor-like proteins. This raises the question of how specific outputs can be generated when receptor proteins are engaged in a plethora of promiscuous interactions. RECEPTOR-LIKE PROTEIN 44 (RLP44) acts to promote both brassinosteroid and phytosulfokine signalling, which orchestrate diverse cellular responses. However, it is unclear how these activities are coordinated. Here, we show that RLP44 is phosphorylated in its highly conserved cytosolic tail and that this post-translational modification governs its subcellular localization. Whereas phosphorylation is essential for brassinosteroid-associated functions of RLP44, its role in phytosulfokine signalling is not affected by phospho-status. Detailed mutational analysis suggests that phospho-charge, rather than modification of individual amino acids determines routing of RLP44 to its target receptor complexes, providing a framework to understand how a common component of different receptor complexes can get specifically engaged in a particular signalling pathway.

14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis

ObjectivesOsteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in...

Immune Gene Expression Profiling in Children and Adults with Acute Myeloid Leukemia Identifies Distinct Phenotypic Patterns

Therapeutic strategies in patients with acute myeloid leukemia (AML) have remained relatively unchanged over the last 30 years. AML is only cured in 35-40% of patients <60 years of age and in 5-15% of patients >60 years of age. Elderly patients who are unfit to receive intensive salvage regimens can only be offered low-intensity therapy or best supportive care. In children, AML accounts for 20% of leukemias. Although intensive multi-agent chemotherapy in conjunction with improved supportive care has increased survival rates to 60-70%, 30-40% of children with AML relapse and only one-third of them will survive to adulthood. The development and delivery of new therapeutic strategies for high-risk AML, including immunotherapy, therefore remains a priority.Tumor phenotypes are dictated not only by the neoplastic cell component, but also by the tumor microenvironment (TME), which includes immune and inflammatory cells and mediators. We have previously shown that the treatment of primary AML cells with interferon (IFN)-γ in vitro induces functional indoleamine 2,3-dioxygenase-1 (IDO1), a molecule with potent immunosuppressive properties, in 50% of unselected cases (‘IFN-γ responders‘). With a median follow-up of 8 years, these IFN-γ responders experienced a significantly shorter event-free survival and overall survival.This multi-institutional study was undertaken to collect comprehensive immune profiles across the biological heterogeneity of childhood and adult AML, with the aim to implement new molecularly targeted agents for patients with specific immunologic subtypes of AML. We employed the nCounter™ system (NanoString Technologies, Seattle, USA), a medium-throughput, fully automated digital platform, to characterize bone marrow (BM) specimens from 70 patients with non-promyelocytic AML (42 children and 28 adults). Ninety BM samples (63 from patients with de novo AML, 18 from patients with AML in complete remission [CR] and 9 from patients with relapsed AML) were analyzed on the nCounter® FLEX platform, using the RNA Pan-Cancer Immune Profiling Panel. This panel detects the expression of genes for 109 cell surface markers on 24 immune cell types, 30 cancer testis antigens, and >500 immune response genes. Transcriptomic data were normalized using the nSolver™ software package (alpha version 4.0). Patient age and status (disease onset, achievement of CR and disease relapse) were selected as covariates.Hierarchical clustering identified patient subgroups with heightened expression of CD8 T-cell, T helper type 1 (Th1)-cell, B-cell and cytotoxicity-related genes, including genes related to natural killer (NK)-cell function, in the leukemia microenvironment (“immune enriched” patients) (Fig. 1A). Interestingly, a select group of patients over-expressed genes associated with monocyte/macrophage, neutrophil and mast cell functions, with low expression levels of T-cell and B-cell genes. AML patients with ‘immune enriched’ gene profiles also expressed CD8A , IFNG , FOXP3 , the T-cell chemoattractants CXCL9 , CXCL10, and inhibitory molecules including IDO1 and the immune checkpoints LAG3 , CTLA4 and PDL1 . This immune landscape is consistent with a ‘T-cell inflamed’ phenotype that might underpin the establishment of adaptive immune resistance and immune escape. Gene set analyses identified differences in immune gene expression levels between childhood and adult AMLs, as well as patients with newly diagnosed AML, relapsed AML or AML in CR (Fig. 1B-C). In particular, genes highly expressed in adult AMLs compared with childhood disease included chemokine, dendritic cell and macrophage genes . In contrast, immune genes down-regulated in adult compared with childhood AMLs included integrin family members, cytokine receptors, T-cell receptor complex components (CD3EAP ) and lipocalin-2 (LCN2 ), a neutrophil gelatinase-associated molecule implicated in suppression of tumor invasiveness and metastasis.In conclusion, our analysis has identified heterogeneous immunological profiles in children and adults with AML at different disease stages. From a clinical standpoint, ‘immune enriched’ AMLs might be amenable to immunotherapy approaches tailored to the BM microenvironment, including blockade of co-inhibitory molecules and/or small-molecule IDO1 inhibitors.Grant support: Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). [Display omitted] DisclosuresWarren:NanoString Technologies: Employment, Equity Ownership. Hood:NanoString Technologies: Employment, Equity Ownership. Danaher:NanoString Technologies: Employment, Equity Ownership. Cesano:NanoString Technologies: Employment, Equity Ownership. Beechem:NanoString Technologies: Employment, Equity Ownership. Tasian:Gilead Sciences, Inc.: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rutella:NanoString Technologies, Seattle, USA: Research Funding.

CYFIP1 Dosages Exhibit Divergent Behavioral Impact via Diametric Regulation of NMDA Receptor Complex Translation in Mouse Models of Psychiatric Disorders

BackgroundGene dosage imbalance caused by copy number variations (CNVs) is a prominent contributor to brain disorders. In particular, 15q11.2 CNV duplications and deletions have been associated with autism spectrum disorder and schizophrenia, respectively. The mechanism underlying these diametric contributions remains unclear. MethodsWe established both loss-of-function and gain-of-function mouse models of Cyfip1, one of four genes within 15q11.2 CNVs. To assess the functional consequences of altered CYFIP1 levels, we performed systematic investigations on behavioral, electrophysiological, and biochemical phenotypes in both mouse models. In addition, we utilized RNA immunoprecipitation sequencing (RIP-seq) analysis to reveal molecular targets of CYFIP1 in vivo. ResultsCyfip1 loss-of-function and gain-of function mouse models exhibited distinct and shared behavioral abnormalities related to autism spectrum disorder and schizophrenia. RIP-seq analysis identified messenger RNA targets of CYFIP1 in vivo, including postsynaptic NMDA receptor (NMDAR) complex components. In addition, these mouse models showed diametric changes in levels of postsynaptic NMDAR complex components at synapses because of dysregulated protein translation, resulting in bidirectional alteration of NMDAR-mediated signaling. Importantly, pharmacological balancing of NMDAR signaling in these mouse models with diametric Cyfip1 dosages rescues behavioral abnormalities. ConclusionsCYFIP1 regulates protein translation of NMDAR and associated complex components at synapses to maintain normal synaptic functions and behaviors. Our integrated analyses provide insight into how gene dosage imbalance caused by CNVs may contribute to divergent neuropsychiatric disorders.

Itch intensity in prurigo nodularis is closely related to dermal interleukin-31, oncostatin M, IL-31 receptor alpha and oncostatin M receptor beta.

Prurigo nodularis (PN) is a chronic skin dermatosis with hyperkeratotic and intensely pruritic nodules. Managing PN-associated itch is difficult because its aetiology is still unknown. This study aimed to investigate the correlation between itch intensity in PN and the expression of a pruritogenic cytokine interleukin (IL)-31, its receptor complex components IL-31 receptor α (IL-31RA) and oncostatin M receptor β (OSMRβ), and oncostatin M (OSM), which is a ligand of OSMR β, through immunofluorescence staining examination. Itch intensity in PN was closely correlated with the number of dermal IL-31(+) cells (Spearman's r=0.551, p<0.05), dermal IL-31RA(+) cells (r=0.475, p<0.05) and dermal OSM(+) cells (r=0.505, p<0.05). In addition, the number of dermal OSMRβ (+) cells was increased in PN (t test, p<0.05), despite not being correlated with itch intensity (Spearman's r=0.375, p>0.05). Major cellular sources of dermal IL-31 were T cells (27.0% of total IL-31-expressing cells) and macrophages (35.0%), while those of OSM were mainly T cells (49.8%) and mast cells (26.8%). IL-31RA-expressing dermal cells were mostly mast cells (49.3%) and macrophages (36.6%), and OSMRβ was mainly expressed by macrophages (51.8%) in the dermis. These findings indicate that IL-31 (mainly from macrophages and T cells) and OSM (principally from T cells and mast cells) stimulate dermal cells expressing IL-31RA and OSMRβ (e.g. macrophages), which may further promote itch and inflammation in PN. This complex dermal milieu of cell/cytokine/receptor network can be a therapeutic target for PN-associated itch.

CD157 and Brain Immune System in (Patho)physiological Conditions: Focus on Brain Plasticity.

Ectoenzyme and receptor BST-1/CD157 has been considered as a key molecule involved in the regulation of functional activity of cells in various tissues and organs. It is commonly accepted that CD157 catalyzes NAD+ hydrolysis and acts as a component of integrin adhesion receptor complex. Such properties are important for the regulatory role of CD157 in neuronal and glial cells: in addition to recently discovered role in the regulation of emotions, motor functions, and social behavior, CD157 might serve as an important component of innate immune reactions in the central nervous system. Activation of innate immune system in the brain occurs in response to infectious agents as well as in brain injury and neurodegeneration. As an example, in microglial cells, association of CD157 with CD11b/CD18 complex drives reactive gliosis and neuroinflammation evident in brain ischemia, chronic neurodegeneration, and aging. There are various non-substrate ligands of CD157 belonging to the family of extracellular matrix proteins (fibronectin, collagen I, finbrinogen, and laminin) whose activity is required for controlling cell adhesion and migration. Therefore, CD157 could control structural and functional integrity of the blood-brain barrier and barriergenesis. On the other hand, contribution of CD157 to the regulation of brain development is rather possible since in the embryonic brain, CD157 expression is very high, whereas in the adult brain, CD157 is expressed on neural stem cells and, presumably, is involved in the neurogenesis. Besides, CD157 could mediate astrocytes’ action on neural stem and progenitor cells within neurogenic niches. In this review we will summarize how CD157 may affect brain plasticity acting as a molecule at the crossroad of neurogenesis, cerebral angiogenesis, and immune regulation.

Gene expression of neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex components in the olfactory organ and brain during seaward and homeward migration by pink salmon (Oncorhynchus gorbuscha).

The expression of synaptic vesicle exocytosis-regulator SNARE complex component genes (snap25, stx1 and vamp2) was examined in the olfactory nervous system during seaward and homeward migration by pink salmon (Oncorhynchus gorbuscha). The expression levels of snares in the olfactory organ were higher in seaward fry than in feeding and homeward adults, reflecting the development of the olfactory nervous system. The expression of snap25a, b and stx1a was upregulated or stable in the adult olfactory bulb and telencephalon. This upregulated expression suggested alterations in olfactory neuronal plasticity that may be related to the discrimination of natal rivers. The expression of stx1b was downregulated in the adult olfactory bulb, but remained stable in the adult telencephalon. The expression of vamp2 was initially strong in seaward fry, but was downregulated in adults in both the olfactory bulb and telencephalon. Pink salmon has the lowest diversity of maturation age, the largest population, and the most evolutional position in Pacific salmon (genus Oncorhynchus). The expression of snares in the olfactory center of pink salmon reflected the timing of sexual maturation and homeward migration. The present results and our previous studies indicate that snares show distinct expression patterns between two salmon species that depend on physiological and ecological features of migration.

Vertebrate insulin alters the expression profile of steroid hormone ecdysone receptor complex components and autophagy-related genes in the pupal fat body of the silkworm, Bombyxmori

Various studies have shown that the insulin-like peptides present in insects and investigation of their possible effects on insect physiology have significance for clarifying the evolutionary developmental period of insulin. The insect fat body is an organ analog to vertebrate adipose tissue and liver. In insects which do not have feeding activity during pupal and adult stages such as Bombyx mori, substances and energy required for continuation of life and development of tissues and organs are provided by the fat body via the autophagy process. In this study, we aimed to analyze the action of mammalian insulin in Bombyx pupae by studying its effects on physiological parameters, and the transcript levels of both ecdysone receptors and autophagy-related genes Atg8 and Atg12. Our results showed that the biochemical contents of the Bombyx mori fat body and the expression of genes related to autophagy and ecdysone receptor complex elements responded differently to insulin treatment depending on the application time.

Functional reconstitution of TatB into the thylakoidal Tat translocase

We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twin-arginine protein transport (Tat) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analysed with the authentic Tat substrate pOEC16, reached routinely 20–25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutated TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional analyses of protein variants. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

Engineering plants to secrete affinity-tagged pathogen elicitors for deciphering immune receptor complex or inducing enhanced immunity.

Plant cells mount plenty of pattern-recognition receptors (PRRs) to detect the microbe-associated molecular patterns (MAMPs) from potential microbial pathogens. MAMPs are overrepresented by proteinaneous patterns, such as the flg22 peptide from bacterial flagellin. Identification of PRR receptor complex components by forward or reverse genetics can be time/labor-consuming, and be confounded by functional redundancies. Here, we present a strategy for identifying PRR complex components by engineering plants to inducibly secrete affinity-tagged proteinaneous MAMPs to the apoplast. The PRR protein complexes bound to self-secreted MAMPs are enriched through affinity purification and dissected by mass spectrometry. As a proof of principle, we could capture the flg22 receptor FLS2 and co-receptor BAK1 using Arabidopsis plants secreting FLAG-tagged flg22 under estradiol induction. Moreover, we identified receptor-like kinases LIK1 and PEPR1/PEPR2 as potential components in the FLS2 receptor complex, which were further validated by protein-protein interaction assays and the reverse genetics approach. Our study showcases a simple way to biochemically identify endogenous PRR complex components without overexpressing the PRR or using chemical cross-linkers, and suggests a possible crosstalk between different immune receptors in plants. A modest dose of estradiol can also be applied to inducing enhanced immunity in engineered plants to both bacterial and fungal pathogens.