Formation Of Signaling Complex Research Articles

The β-arrestin1/endothelin axis bolsters ovarian fibroblast-dependent invadosome activity and cancer cell metastatic potential

Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ETA) and B (ETB) receptors, ET-1 enables the recruitment of β-arrestin1 (β-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might “educate” human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETA/BR/β-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin β1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETA/BR or β-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETA/BR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/β-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETA/BR antagonists.

Diversification of Toll-like receptor 1 in swamp eel (Monopterus albus)

Toll-like receptor 1 (TLR1) is a pattern recognition receptor that plays critical roles in triggering immune activation via detecting bacterial lipoproteins and lipopeptides. In this study, the genetic characteristic of TLR1 was studied for an important aquaculture fish, swamp eel Monopterus albus. The eel has been seriously threatened by infectious diseases. However, a low level of genetic heterogeneity in the fish that has resulted from a demographic bottleneck presents further challenges in breeding for disease resistance. A comparison with the homologue of closely related species M. javanensis revealed that amino acid replacement (nonsynonymous) but not silent (synonymous) differences have accumulated nonrandomly over the coding sequences of the receptors at the early stage of their phylogenetic split. The combined results from comparative analyses of nonsynonymous-to-synonymous polymorphisms showed that the receptor has undergone significant diversification in M. albus driven by adaptive selection likely after the genetic bottleneck. Some of the changes reported here have taken place in the structures mediating heterodimerization with co-receptor TLR2, ligand recognition, and/or formation of active signaling complex with adaptor, which highlighted key structural elements and strategies of TLR1 in arms race against exogenous challenges. The findings of this study will add to the knowledge base of genetic engineering and breeding for disease resistance in the eel.

Glutamine-mediated epigenetic regulation of cFLIP underlies resistance to TRAIL in pancreatic cancer

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it kills cancer cells while sparing normal cells. However, many cancers, including pancreatic ductal adenocarcinoma (PDAC), exhibit intrinsic or acquired resistance to TRAIL, and the molecular mechanisms underlying TRAIL resistance in cancers, particularly in PDAC, remain unclear. In this study, we demonstrated that glutamine (Gln) endows PDAC cells with resistance to TRAIL through KDM4C-mediated epigenetic regulation of cFLIP. Inhibition of glutaminolysis significantly reduced the cFLIP level, leading to TRAIL-mediated formation of death-inducing signaling complexes. Overexpression of cFLIP dramatically rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Alpha-Ketoglutarate (aKG) supplementation significantly reversed the decrease in the cFLIP level induced by glutaminolysis inhibition and rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Knockdown of glutamic-oxaloacetic transaminase 2, which facilitates the conversion of oxaloacetate and glutamate into aspartate and aKG, decreased aKG production and the cFLIP level and activated TRAIL-induced apoptosis. AKG-mediated epigenetic regulation was necessary for maintaining a high level of cFLIP. Glutaminolysis inhibition increased the abundance of H3K9me3 in the cFLIP promoter, indicating that Gln-derived aKG production is important for Jumonji-domain histone demethylase (JHDM)-mediated cFLIP regulation. The JHDM KDM4C regulated cFLIP expression by binding to its promoter, and KDM4C knockdown sensitized PDAC cells to TRAIL-induced apoptosis. The present findings suggest that Gln-derived aKG production is required for KDM4C-mediated epigenetic regulation of cFLIP, which leads to resistance to TRAIL.

Abstract 2460: Receptor antibody conjugated engager (RACE): A novel bispecific 4-1BB engager platform for enhanced immuno-oncology therapy

Abstract Background: Traditional immuno-oncology strategies, such as checkpoint blockade therapy, are evolving to immune cell engagers as a smarter strategy. The early successes of Blincyto, a CD3 engager, demonstrated its capacity to activate host pan-T cells irrespective of their TCR-pMHC restriction status. However, this broad activation has been linked to off-target immunotoxicities, thus limiting its therapeutic applicability. In response, targeting immune co-stimulatory receptors like CD28 or 4-1BB has gained attention by their expression being induced on activated immune effector cells, presenting a more targeted approach to cancer therapy. Methods: We developed RACE, a novel bispecific 4-1BB engager. This platform fuses a 4-1BB ligand (4-1BBL) trimer with antibody F(ab’)2 fragments, excluding Fc domains. A Key and lock design on the 4-1BBL trimer interface facilitates asymmetric assembly of 4-1BBL dimer and monomer in the endoplasmic reticulum, achieving over 95% assembly efficiency in mammalian systems. Results: RACE differs from conventional 4-1BB agonistic antibodies in its ability to form natural hexagonal hyper-clusters of 4-1BB receptors, enhancing NF-κB signal transduction. Unlike first-generation 4-1BB antibodies, which incurred off-tumor hepatotoxicity, RACE selectively activates 4-1BB receptors only in the presence of tumor antigens. Enhanced cytotoxicity requires successful TCR signaling complex formation between T cells and cancer cells. In CT26 and MC38 tumor models, RACE not only induced strong anti-tumor effects without hepatic or systemic toxicities but showed synergistic efficacy with anti-PD-1 antibodies as well. Conclusion: The novel RACE platform represents a potent as well as safe immuno-oncology strategy, demonstrating significant potential for clinical application. Citation Format: DaeHee Lee, Jihye Yoon, Jonggwan Jeong, Jeonghyeon Bak, KkaBi Son, Heechan Kim, Seil Jang, Minseok S. Kim. Receptor antibody conjugated engager (RACE): A novel bispecific 4-1BB engager platform for enhanced immuno-oncology therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2460.

An optogenetic method for the controlled release of single molecules

We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

An inducible Cd79b mutation confers ibrutinib sensitivity in mouse models of Myd88-driven diffuse large B-cell lymphoma

AbstractDiffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma and constitutes a highly heterogenous disease. Recent comprehensive genomic profiling revealed the identity of numerous molecularly defined DLBCL subtypes, including a cluster which is characterized by recurrent aberrations in MYD88, CD79B, and BCL2, as well as various lesions promoting a block in plasma cell differentiation, including PRDM1, TBL1XR1, and SPIB. Here, we generated a series of autochthonous mouse models to mimic this DLBCL cluster and specifically focused on the impact of Cd79b mutations in this setting. We show that canonical Cd79b immunoreceptor tyrosine-based activation motif (ITAM) mutations do not accelerate Myd88- and BCL2-driven lymphomagenesis. Cd79b-mutant murine DLBCL were enriched for IgM surface expression, reminiscent of their human counterparts. Moreover, Cd79b-mutant lymphomas displayed a robust formation of cytoplasmic signaling complexes involving MYD88, CD79B, MALT1, and BTK. These complexes were disrupted upon pharmacological BTK inhibition. The BTK inhibitor–mediated disruption of these signaling complexes translated into a selective ibrutinib sensitivity of lymphomas harboring combined Cd79b and Myd88 mutations. Altogether, this in-depth cross-species comparison provides a framework for the development of molecularly targeted therapeutic intervention strategies in DLBCL.

Isothermal Titration Calorimetry to Study Plant Peptide Ligand-Receptor Interactions.

The field of plant receptor biology has rapidly expanded in the past three decades. However, the demonstration of direct interaction between receptor-ligand pairs remains a challenge. Identifying and quantifying protein-ligand interactions is crucial for understanding how they regulate certain physiological processes. An important aspect is the quantification of different parameters of the interaction, like binding affinity, kinetics, and ligand specificity that drive the formation of signaling complexes. In this chapter, we discuss Isothermal Titration Calorimetry (ITC) as a label-free technique to measure thermodynamic parameters of ligand binding with high accuracy and reproducibility. We provide a detailed guideline how to design, perform, analyze, and interpret ITC measurements using as an example the interaction between the SCHENGEN3/GASSHO1 (SGN3/GSO1) leucine-rich repeat receptor-like kinase and its sulfated peptide ligand CASPARIAN STRIP INTEGRITY FACTOR 2 (CIF2).

An In VivoPiggyBac Insertional Mutagenesis Screen Reveals Oncogenic Lesions Cooperating with Myd88 L265P

Diffuse large B cell lymphoma (DLBCL) is the most common Non-Hodgkin lymphoma and originates from transformed germinal center-experienced B cells. Traditionally, DLBCL has been divided into two subtypes, depending on whether the transcriptional profile of the tumor relates to an activated B cell or a germinal center B cell (ABC and GCB DLBCL, Alizadeh et al., 2000). More recent efforts classified DLBCL cases based on their genetic aberrations and identified several clusters with distinct mutational profiles (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020). The MCD/C5 cluster is characterized by recurrent mutations in MYD88, PRDM1 and frequent amplifications of BCL2, amongst others. We recently showed that mice harboring a B cell-specific Myd88 L252P mutation (orthologous position of the human p.L265P mutation) develop B cell proliferation and occasional transformation into DLBCL (Knittel et al., 2016). Lymphomagenesis is further increased when Myd88 L252P is combined with BCL2 overexpression and a genetically engineered block in plasmacytic differentiation by loss of Prdm1 or overexpression of Spib (Flümann et al., 2021). To ask which further genes and pathways cooperate with Myd88 L252P in lymphomagenesis, we performed an in vivo piggyBac insertional mutagenesis screen. In this system, a conditionally expressed transposase mobilizes transposable elements. These elements can then reintegrate into the genome and either silence or drive the expression of genes, depending on the exact integration site and orientation of the transposon cassette (Rad et al., 2015). We crossed this piggyBac system onto a Myd88 L252P background to identify genes that cooperate with Myd88 mutations in lymphomagenesis. Mice harboring both the Myd88 mutant allele and the piggyBac system lived significantly shorter than controls harboring only the Myd88 mutation or just the piggyBac system. Myd88/piggyBac animals developed B220 +/CD138 - lymphomas and DNA isolated from these lesions allowed the detection of common transposon insertion sites. Among the genes significantly enriched for integrations, we observed known genetic drivers of human MCD/C5 DLBCL, such as PIM1 and ETV6. We also identified TBL1XR1 and SPIB as common insertion sites, as well as BCL2, BIM1 and BCL-XL, further validating our approach of engineering a plasma cell differentiation block as well as anti-apoptotic BCL2 overexpression on a Myd88-mutant background to model MCD DLBCL. Of note, the hits identified in this screen were significantly distinct from hits identified to drive Myc-driven B cell lymphomagenesis (Weber et al., 2019). Additionally, we identified several candidate genes that are not reported to be frequently genetically altered in DLBCL, however associated with processes relevant to B cell biology, particularly to B cell receptor signaling. As ‘B cell receptor signaling’ was a prominent term in our screen hits and CD79B ITAM mutations are an additional hallmark of MCD/C5 DLBCL (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020), we introduced a conditional Cd79b p.Y195H allele to our Cd19 Cre/wt;Myd88 cond.p.L252P/wt; Rosa26 LSL.BCL2-IRES-GFP/wt; Prdm1 fl/fl MCD/C5 DLBCL model. While we did not observe significant differences in the immunohistochemial and transcriptional phenotype of Cd79b WT and mutant lymphomas, Cd79b p.Y195H tumors showed increased B cell receptor (BCR) signaling activity, indicated by increased levels of phosphorylated SYK and PLCg2. Cd79b mutant lymphomas also showed an increased formation of cytoplasmic signaling complexes comprised of MYD88 and several components of the BCR pathway, including MALT1 and BTK. The formation of these complexes depended on active BTK, as treatment with the BTK inhibitor ibrutinib reduced complex formation to levels found in Cd79b WT lymphomas. Consequently, we investigated the effects of ibrutinib treatment in Cd79b-mutant and wildtype MCD DLBCL mouse models and found Cd79b-mutant lymphomas to be significantly more sensitive to ibrutinib treatment than their Cd79b WT counterparts.

Abstract P1163: Identifying Novel Intracellular Interactions Driving Membrane-bound Tumour Necrosis Factor-mediated Signalling In The Microvasculature

Elevated total peripheral resistance (TPR) is a hallmark of many cardiovascular diseases and furthers disease progression. We previously demonstrated that myogenic tone, regulated by membrane-bound tumour necrosis factor (mTNF) reverse signalling, is a primary modulator of TPR. Thus, inhibiting mTNF reverse signalling should reduce TPR with the potential to improve disease outcome. However, since mTNF is a ubiquitous protein with critical biological functions, its indiscriminate inhibition can cause widespread side effects. Therefore, therapeutics aiming to reduce myogenic tone and TPR must target other elements of mTNF reverse signalling. To identify these unknown participants of mTNF reverse signalling, we used proximity-dependent biotinylation coupled to mass spectrometry (BioID). Our search identified 42 high confidence hits that interact with and/or are proximal to mTNF’s cytoplasmic domain. Gene ontology (GO) analysis revealed that molecularly, these proteins are structural constituents of the cytoskeleton involved in actin and cell adhesion molecule binding. This aligns with our underlying hypothesis that mTNF functions as a mechanosensor that tethers to its receptors and initiates myogenic vasoconstriction in response to changes in transmural pressure. GO also identified proteins involved in MAPK and RhoA signalling, which are critically involved in myogenic vasoconstriction. Our most abundant hit (fold change = 12.6, p-value = 0.0023) is a cytoskeletal protein that plays a role in maintaining the structural integrity of cells. This protein also interacts with proteins involved in various signalling cascades, namely, RhoA and calcium-dependent signalling pathways. Thus, we hypothesize that this protein is essential for formation of signalling complexes with mTNF and interacts with other proteins to mediate reverse signalling . We will employ a bioluminescence-based approach to quantify the strength of all interactions detected by BioID and identify small molecule inhibitors that can disrupt these interactions. These newly discovered inhibitors could potentially be used as novel therapeutics targeting myogenic tone and hence, elevated TPR to improve disease management and clinical outcome of cardiovascular diseases.

Biomarkers derived from CmP signal network in triple negative breast cancers.

Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer-related death in women, accounting for approximately 30% of all new cancer cases. The prognosis of breast cancer heavily depends on the stage of diagnosis, with early detection resulting in higher survival rates. Various risk factors, including family history, alcohol consumption and hormone exposure, contribute to breast cancer development. Triple-negative breast cancer (TNBC), characterized by the absence of certain receptors, is particularly aggressive and heterogeneous. Cerebral cavernous malformations (CCMs), abnormal dilations of small blood vessels in the brain, is contributed by mutated genes like CCM1, CCM2, and CCM3 through the perturbed formation of the CCM signaling complex (CSC). The CSC-non-classic membrane progesterone receptors (mPRs)-progesterone (PRG) (CmP)/CSC-mPRs-PRG-classic nuclear progesterone receptors (nPRs) (CmPn) signaling network, which integrates the CSC with mPRs and nPRs, plays a role in breast cancer tumorigenesis. Understanding these pathways can provide insights into potential treatments. This paper focuses on the emerging field of CmPn/CmP signal networks, which involve PRG, its receptors (nPRs and mPRs), and the CSC. These networks play a role in tumorigenesis, particularly in TNBCs. Aims to deliver a thorough examination of the CmP/CmPn pathways concerning TNBCs, this paper provides a comprehensive overview of these pathways, explores their applications and highlights their significance in the context of TNBCs.

HPIP is an essential scaffolding protein running through the EGFR-RAS-ERK pathway and drives tumorigenesis.

The EGFR-RAS-ERK pathway plays a key role in cancer development and progression. However, the integral assembly of EGFR-RAS-ERK signaling complexes from the upstream component EGFR to the downstream component ERK is largely unknown. Here, we show that hematopoietic PBX-interacting protein (HPIP) interacts with all classical components of the EGFR-RAS-ERK pathway and forms at least two complexes with overlapping components. Experiments of HPIP knockout or knockdown and chemical inhibition of HPIP expression showed that HPIP is required for EGFR-RAS-ERK signaling complex formation, EGFR-RAS-ERK signaling activation, and EGFR-RAS-ERK signaling-mediated promotion of aerobic glycolysis as well as cancer cell growth in vitro and in vivo. HPIP expression is correlated with EGFR-RAS-ERK signaling activation and predicts worse clinical outcomes in patients with lung cancer. These results provide insights into EGFR-RAS-ERK signaling complex formation and EGFR-RAS-ERK signaling regulation and suggest that HPIP may be a promising therapeutic target for cancer with dysregulated EGFR-RAS-ERK signaling.

Anionic phospholipids control mechanisms of GPCR-G protein recognition

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

Biophysics of IL-22 receptor interaction with selected interleukins.

Atopic dermatitis (AD) is the most prevalent chronic inflammatory skin condition globally, and many patients do not respond adequately to currently available therapies [1]. Interleukin-22 receptor 1 (IL-22RA1) has been identified as a potential mediator of AD, but no treatments for this pathway are currently on the market [2]. In this work, we aim to elucidate the biophysics of the binary and ternary IL-22RA1 complex formation with interleukins by combining surface plasmon resonance requiring protein immobilization and in-solution techniques such as small angle X-ray scattering and circular dichroism. In addition, structural characterization of the ternary complexes by high-resolution cryogenic electron microscopy is ongoing and will be used to provide Angstrom resolution structural data about the complexes. This will be coupled to molecular dynamics simulations, providing a comprehensive overview of the formation of IL-22RA1 signaling complexes resulting in knowledge on the domains involved in the signaling process. This will result in new and novel knowledge on the interaction with these interleukins and IL22R1 and provide insights into the role of these interleukins and potential approaches to block their interaction by an antibody approach.

Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR.

G protein-coupled receptor (GPCR) conformational plasticity enables formation of ternary signaling complexes with intracellular proteins in response to binding extracellular ligands. We investigate the dynamic process of GPCR complex formation in solution with the human A2A adenosine receptor (A2AAR) and an engineered Gs protein, mini-Gs. 2D nuclear magnetic resonance (NMR) data with uniform stable isotope-labeled A2AAR enabled a global comparison of A2AAR conformations between complexes with an agonist and mini-Gs and with an agonist alone. The two conformations are similar and show subtle differences at the receptor intracellular surface, supporting a model whereby agonist binding alone is sufficient to populate a conformation resembling the active state. However, an A2AAR "hot spot" connecting the extracellular ligand-binding pocket to the intracellular surface is observed to be highly dynamic in the ternary complex, suggesting a mechanism for allosteric connection between the bound G protein and the drug-binding pocket involving structural plasticity of the "toggle switch" tryptophan.

Pim1 promotes IFN-β production by interacting with IRF3

The Pim (proviral integration site for Moloney murine leukemia virus) proteins compose a serine threonine kinase family whose members regulate cell proliferation, migration and cell survival. However, whether Pim kinases participate in innate immune responses is unclear. Here, we show for the first time that Pim1 plays an essential role in the production of interferon (IFN)-β by macrophages after their Toll-like receptor (TLR) pathway is activated by pathogen-associated molecular patterns (PAMPs). Specifically, Pim1 was quickly upregulated in an NF-κB-dependent manner after TLR stimulation with PAMPs. Pim1 deficiency reduced TLR3- or TLR4-stimulated IFN-β and IFN-stimulated gene (ISG) expression but not proinflammatory cytokine expression in macrophages. Mechanistically, Pim1 specifically upregulates IRF3 phosphorylation and nuclear translocation. However, this role is not dependent on Pim1 kinase activity. Rather, Pim1 appears to promote IRF3 phosphorylation by enhancing the formation of IFN-β signaling complexes composed of TRIF, TRAF3, TBK1, and IRF3. Poly (I:C)-treated Pim1−/− mice produced less serum IFN-β and were less likely to survive than wild-type mice. These findings show for the first time that Pim1 participates in TLR-mediated IFN-β production, thus revealing a novel target for controlling antiviral innate immune responses.

Rubiarbonol B induces RIPK1-dependent necroptosis via NOX1-derived ROS production.

The activation of receptor-interacting protein kinase 1 (RIPK1) by death-inducing signaling complex (DISC) formation is essential for triggering the necroptotic mode of cell death under apoptosis-deficient conditions. Thus, targeting the induction of necroptosis by modulating RIPK1 activity could be an effective strategy to bypass apoptosis resistance in certain types of cancer. In this study, we screened a series of arborinane triterpenoids purified from Rubia philippinesis and identified rubiarbonol B (Ru-B) as a potent caspase-8 activator that induces DISC-mediated apoptosis in multiple types of cancer cells. However, in RIPK3-expressing human colorectal cancer (CRC) cells, the pharmacological or genetic inhibition of caspase-8 shifted the mode of cell death by Ru-B from apoptosis to necroptosis though upregulation of RIPK1 phosphorylation. Conversely, Ru-B-induced cell death was almost completely abrogated by RIPK1 deficiency. The enhanced RIPK1 phosphorylation and necroptosis triggered by Ru-B treatment occurred independently of tumor necrosis factor receptor signaling and was mediated by the production of reactive oxygen species via NADPH oxidase 1 in CRC cells. Thus, we propose Ru-B as a novel anticancer agent that activates RIPK1-dependent cell death via ROS production, and suggest its potential as a novel necroptosis-targeting compound in apoptosis-resistant CRC.

Epithelial JAM-A is fundamental for intestinal wound repair in vivo.

Junctional adhesion molecule-A (JAM-A) is expressed in several cell types, including epithelial and endothelial cells, as well as some leukocytes. In intestinal epithelial cells (IEC), JAM-A localizes to cell junctions and plays a role in regulating barrier function. In vitro studies with model cell lines have shown that JAM-A contributes to IEC migration; however, in vivo studies investigating the role of JAM-A in cell migration–dependent processes such as mucosal wound repair have not been performed. In this study, we developed an inducible intestinal epithelial–specific JAM-A–knockdown mouse model (Jam-aERΔIEC). While acute induction of IEC-specific loss of JAM-A did not result in spontaneous colitis, such mice had significantly impaired mucosal healing after chemically induced colitis and after biopsy colonic wounding. In vitro primary cultures of JAM-A–deficient IEC demonstrated impaired migration in wound healing assays. Mechanistic studies revealed that JAM-A stabilizes formation of protein signaling complexes containing Rap1A/Talin/β1 integrin at focal adhesions of migrating IECs. Loss of JAM-A in primary IEC led to decreased Rap1A activity and protein levels of Talin and β1 integrin, and it led to a reduction in focal adhesion structures. These findings suggest that epithelial JAM-A plays a critical role in controlling mucosal repair in vivo through dynamic regulation of focal adhesions.

The guidance and adhesion protein FLRT2 dimerizes in cis via dual small-X3-small transmembrane motifs

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.