Membrane-bound Receptors Research Articles

SPION decorated exosome delivery of TNF-α to cancer cell membranes through magnetism.

Tumor necrosis factor (TNF-α) is capable of inducing apoptosis and is a promising candidate for genetic engineering drugs in cancer therapy; however, the serious side-effects of TNF-α hinder their clinical application. In the present study, a method for preparing fusion proteins of cell-penetrating peptides (CPP) and TNF-α (CTNF-α)-anchored exosomes coupled with superparamagnetic iron oxide nanoparticles (CTNF-α-exosome-SPIONs) with membrane targeting anticancer activity has been demonstrated. To acquire exosomes with TNF-α anchored in its membrane, a CTNF-α expression vector was constructed and a stable mesenchymal stem cell cell line that expressed CTNF-α was established. Conjugating transferrin-modified SPIONs (Tf-SPIONs) onto CTNF-α-exosomes through transferrin-transferrin receptor (Tf-TfR) interaction yields CTNF-α-exosome-SPIONs with good water dispersibility. The incorporation of TNF-α into exosomes and the conjugation of SPIONs significantly enhanced the binding capacity of TNF-α to its membrane-bound receptor TNFR I, thus increasing the therapeutic effects. CTNF-α-exosome-SPIONs significantly enhanced tumor cell growth inhibition via induction of the TNFR I-mediated apoptotic pathway. In vivo studies using murine melanoma subcutaneous cancer models showed that TNF-α-loaded exosome-based vehicle delivery enhanced cancer targeting under an external magnetic field and suppressed tumor growth with mitigating toxicity. Taken together, our results suggest that CTNF-α-exosome-SPIONs showed great potential in membrane targeting therapy.

Co-Expression Profile of TNF Membrane-Bound Receptors Type 1 and 2 in Rheumatoid Arthritis on Immunocompetent Cells Subsets.

Introduction: Tumor necrosis factor (TNFα) is an important proinflammatory cytokine in rheumatoid arthritis (RA) immune processes. However, TNFα activity and functions may be regulated by soluble receptors, which act as decoys, and by number, density, and co-expression of its membrane-bound receptors type 1 and 2 (TNFR1 and TNFR2). The aim of this study was to reveal associations between TNFR1/2 co-expression profile parameters and RA disease activity indicators. Methods: PBMC were analyzed from 46 healthy donors and 64 patients with RA using flow cytometry. Patients were divided according to the disease activity score (DAS) 28 index into groups with high (n = 22, 34.4%), moderate (n = 30, 46.9%), and low (n = 12, 18.8%) disease activity. Co-expression of TNFR1 and TNFR2 was studied by evaluating the percentage of cells, with different receptors, and by counting the number of receptors of each type per cell, using QuantiBritePE beads. Associations between disease severity and activity indicators and parameters of TNFα receptor expression in subpopulations of immune cells were studied. Results: T cell subsets from RA patients were characterized by co-expression of TNFR1 and TNFR2, and were found to differ significantly compared with healthy donors. Memory cells both among T helper cells and cytotoxic T cells demonstrated the most significant differences in TNFR-expression profile. Multivariable logistic regression revealed model to identified RA patients from healthy individual based on the TNFR1/2 co-expression parameters. Conclusion: The profile of TNFR1\\2 co-expression differs in RA comparing with health. Proportion of TNFR1+TNFR2- cells increased significantly among memory T helper cells and activated cytotoxic T cells, and decreased significantly among naïve cytotoxic T cells and T regulatory cells as compared with health. The parameters of TNFR1\\2 co-expression in RA are associated with clinical and laboratory indicators of disease activity.

P2 receptors mRNA expression profiles in macrophages from ankylosing spondylitis patients and healthy individuals.

Ankylosing spondylitis (AS) is a multifactorial rheumatic disease which mainly involves the axial skeleton. Macrophages and extracellular nucleotides have been shown to contribute to the inflammation process in autoimmune diseases. Membrane-bound purinergic P2 receptors might be involved in the modulation of immune cells in AS. Therefore, we aimed to analyze the messenger RNA (mRNA) expression of P2 receptors in the macrophages of AS patients and healthy controls. Twenty-three AS patients and 23 age- and sex-matched healthy individuals were included in our study. Whole blood-separated monocytes of study participants were stimulated by macrophage colony-stimulating factor for 7days and differentiated to macrophages. Monocyte and macrophage markers were analyzed by flow cytometry. SYBR green real-time polymerase chain reaction was used to measure the relative expression levels of P2RX1 , P2RX2 , P2RX3 , P2RX4 , P2RX5 , P2RX6 , P2RX7 , P2RY1 , P2RY2 , P2RY4 , P2RY6 , P2RY11 , P2RY12 , P2RY13 , P2RY14 , and PANX1 genes. P2RY13 and P2RY6 genes had the highest expression levels in macrophagesamong P2RY genes. P2RY1 mRNA expression was significantly down-regulated (-1.75 fold) and P2RY14 was up-regulated (2.6 fold) in macrophages of AS patients compared to healthy individuals. P2RX4 gene had the highest expression in monocyte-derived macrophages, followed by P2RX7 andP2RX1 genes. There was no significant difference in P2X receptor mRNA expression level between macrophages of AS patients and healthy individuals. Our results indicate that AS patients show altered expression levels of P2 receptor genes. Moreover, these changes might be associated with disease activity and patients' status.

Teleost IgD+IgM− B Cells Mount Clonally Expanded and Mildly Mutated Intestinal IgD Responses in the Absence of Lymphoid Follicles

SummaryImmunoglobulin D (IgD) is an ancient antibody with dual membrane-bound and fluid-phase antigen receptor functions. The biology of secreted IgD remains elusive. Here, we demonstrate that teleost IgD+IgM− plasmablasts constitute a major lymphocyte population in some mucosal surfaces, including the gut mucosa. Remarkably, secreted IgD binds to gut commensal bacteria, which in turn stimulate IgD gene transcription in gut B cells. Accordingly, secreted IgD from gut as well as gill mucosae, but not the spleen, show a V(D)J gene configuration consistent with microbiota-driven clonal expansion and diversification, including mild somatic hypermutation. By showing that secreted IgD establishes a mutualistic relationship with commensals, our findings suggest that secreted IgD may play an evolutionary conserved role in mucosal homeostasis.

Budesonide enhances agonist-induced bronchodilation in human small airways by increasing cAMP production in airway smooth muscle.

The nongenomic mechanisms by which glucocorticoids modulate β2 agonist-induced-bronchodilation remain elusive. Our studies aimed to elucidate mechanisms mediating the beneficial effects of glucocorticoids on agonist-induced bronchodilation. Utilizing human precision-cut lung slices (hPCLS), we measured bronchodilation to formoterol, prostaglandin E2 (PGE2), cholera toxin (CTX), or forskolin in the presence and absence of budesonide. Using cultured human airway smooth muscle (HASM), intracellular cAMP was measured in live cells following exposure to formoterol, PGE2, or forskolin in the presence or absence of budesonide. We showed that simultaneous budesonide administration amplified formoterol-induced bronchodilation and attenuated agonist-induced phosphorylation of myosin light chain, a necessary signaling event mediating force generation. In parallel studies, cAMP levels were augmented by simultaneous exposure of HASM cells to formoterol and budesonide. Budesonide, fluticasone, and prednisone alone rapidly increased cAMP levels, but steroids alone had little effect on bronchodilation in hPCLS. Bronchodilation induced by PGE2, CTX, or forskolin was also augmented by simultaneous exposure to budesonide in hPCLS. Furthermore, HASM cells expressed membrane-bound glucocorticoid receptors that failed to translocate with glucocorticoid stimulation and that potentially mediated the rapid effects of steroids on β2 agonist-induced bronchodilation. Knockdown of glucocorticoid receptor-α had little effect on budesonide-induced and steroid-dependent augmentation of formoterol-induced cAMP generation in HASM. Collectively, these studies suggest that glucocorticoids amplify cAMP-dependent bronchodilation by directly increasing cAMP levels. These studies identify a molecular mechanism by which the combination of glucocorticoids and β2 agonists may augment bronchodilation in diseases such as asthma or chronic obstructive pulmonary disease.

Abstract NT-089: TRAIL-EXPRESSING ONCOLYTIC VACCINIA VIRUS COMBINED WITH SMALL-MOLECULE DRUG PAC1 IS A POTENTIALLY EFFECTIVE TREATMENT ALTERNATIVE FOR OVARIAN CANCERS

Abstract INTRODUCTION: Procaspase activating compound-1 (PAC1) is a small-molecule drug shown in vitro to sequester inhibitory zinc ions from caspase-3. TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic ligand that binds membrane-bound death receptors, triggering the extrinsic apoptotic pathway. Both agents display low toxicity in humans. Vaccinia virus (VACV) is a double-stranded DNA virus that has shown therapeutic efficacy in clinical trials and has an established safety profile in humans due to its use as the smallpox vaccine. First-line standard of care for ovarian cancer is combination taxane and carboplatin, which has significant potential toxicity and 70% of women who receive it suffer relapse. Clinical trials involving TRAIL, both alone and combined with other drugs, have shown that while it is well-tolerated it is ineffective partly due to insufficient dosing at the tumour site. In an effort to uncover a safer, more effective therapeutic for ovarian cancer we report here on the successful construction of a recombinant oncolytic vaccinia virus expressing TRAIL (VACVTRAIL). Secretion of TRAIL by VACV-infected cancer cells will result in localized administration of TRAIL at higher dosages and minimize potential side-effects. We posit that treatment with PAC1 and VACVTRAIL represents a potentially safe, effective treatment for ovarian cancers. RESULTS: Testing in cell line models of granulosa cell tumour (GCT) have shown that recombinant human (rh)TRAIL is effective in combination with PAC1. Dose-response assays established that combination of PAC1 (20 μM) with rhTRAIL (10 ng/mL) dramatically reduced viability of cancer cells while being substantially less toxic to normal cells. Replication of those assays on patient-derived primary and recurrent GCT cells confirmed PAC1 combined with rhTRAIL was dramatically more cytotoxic than treatment with rhTRAIL or PAC1 alone. To optimize delivery of TRAIL to tumour cells, we constructed a recombinant VACVTRAIL virus that secretes TRAIL in the range of 70–80 ng/mL. Dose-response curves showed VACVTRAIL to be strongly cytotoxic with an ED50 of 0.1 plaque forming unit (PFU) per cell. Comparing toxicity of VACVTRAIL to a non-TRAIL-expressing VACV established that secretion of TRAIL is the basis for VACVTRAIL superiority in killing GCT cells, and supernatant collected from infected cells is more effective at reducing cell viability when combined with PAC1 than is rhTRAIL combined with PAC1. CONCLUSION: We have successfully constructed a TRAIL-expressing oncolytic VACV which produces effective levels of active TRAIL from infected cells. Results in vitro suggest combining PAC1 with oncolytic VACVTRAIL will allow localized delivery of TRAIL resulting in a safe, synergistic, self-amplifying therapy. Citation Format: Powel Crosley, Kate Agopsowicz, Kyle Potts, Ryan Noyce, Marjut Pihlajoki, Markku Heikinheimo, Anniina Färkkilä, David Evans, Mary Hitt. TRAIL-EXPRESSING ONCOLYTIC VACCINIA VIRUS COMBINED WITH SMALL-MOLECULE DRUG PAC1 IS A POTENTIALLY EFFECTIVE TREATMENT ALTERNATIVE FOR OVARIAN CANCERS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-089.

IL-7/IL-7R gene variants impact circulating IL-7/IL-7R homeostasis and ART-associated immune recovery status

A relationship between polymorphisms in genes encoding interleukin 7 (IL-7) and its cellular receptor (IL-7R) and antiretroviral therapy (ART)-associated immune recovery in HIV subjects has been previously reported. However, details of this relationship remain unclear, and the association of these polymorphisms with circulating IL-7/IL-7R levels is scarce. Here, we explored whether IL-7/IL-7R axis was associated with quantitative CD4+ T-cell recovery in HIV-infected subjects. IL-7/IL-7R polymorphisms were assessed by genotyping, and multiple inheritance models were used to estimate both, their association with low pre-ART CD4+ T-cell counts and incomplete immune recovery status after 48 weeks of suppressive ART. Integrated data from genetic variants association and soluble plasma IL-7/IL-7R quantification suggest that IL-7/IL-7R genotype expression could alter the homeostatic balance between soluble and membrane-bound receptors. The haplotype analyses indicates that allele combinations impacts pre-ART circulating CD4+ T-cell counts, immune recovery status and the absolute increment of CD4+ T-cell counts. The knowledge about how IL-7/IL-7R axis is related to quantitative CD4+ T-cell recovery and immune recovery status after initiating ART could be useful regarding T-cell reservoirs investigations in HIV subjects.

Regulation of Fibrotic Processes in the Liver by ADAM Proteases.

Fibrosis in the liver is mainly associated with the activation of hepatic stellate cells (HSCs). Both activation and clearance of HSCs can be mediated by ligand–receptor interactions. Members of the a disintegrin and metalloprotease (ADAM) family are involved in the proteolytic release of membrane-bound ligands and receptor ectodomains and the remodelling of the extracellular matrix. ADAM proteases are therefore major regulators of intercellular signalling pathways. In the present review we discuss how ADAM proteases modulate pro- and anti-fibrotic processes and how ADAM proteases might be harnessed therapeutically in the future.

ER-mediated anti-tumor effects of shikonin on breast cancer

Estrogen receptor (ER) is expressed in most Breast cancer (BC) patients. G protein-coupled estrogen receptor (GPER), which is a membrane-bound estrogen receptor, is associated with the tumor development and progression in BC. Shikonin (SK) is a natural compound that is known to have anti-tumor effects. This study aims to assess the effects of shikonin on the cell proliferation, cell cycle and cell apoptosis of BC and whether the effects are related to ER/GPER signaling pathway. The results demonstrated that shikonin inhibited the cellular proliferation of MCF-7 BC cells via G0/G1 arrest and apoptosis in concentration-dependent manner. The anti-proliferative effect of SK on SK-BR-3 BC cells was associated with apoptosis. Both ERα and GPER were expressed in MCF-7 cells, while ERα were negative and GPER were positive in SK-BR-3 cells. Furthermore, shikonin downregulated the expression of ERα and GPER, and this effect was not affected by the estrogen environment. In addition, shikonin downregulated the EGFR and p-ERK expression in MCF-7 and SK-BR-3, which was also not affected by the estrogen environment. EGFR and p-ERK were still suppressed by co-treatment with the selective GPER against G1 or antagonist G15. In conclusion, these results suggest that shikonin shows anti-tumor effects on MCF-7 and SK-BR-3 cells. The effects seem to be associated with EGFR/p-ERK downregulation via ERα and GPER inhibition.

The Progress of Investigating the CD137-CD137L Axis as a Potential Target for Systemic Lupus Erythematosus.

Costimulatory molecules facilitate cross-talks among leukocytes via mutual stimulatory and inhibitory signalling, contributing to diverse immunological outcomes in normal physiological responses and pathological conditions. Systemic lupus erythematosus (SLE) is a complex multi-systemic autoimmune condition in which cellular communication through the involvement of costimulatory molecules is crucial in driving proinflammatory responses from the stage of autoantigen presentation to the subsequent process of pathogenic autoantibody production. While the physiology of the costimulatory systems including OX40-OX40L, CD28/CTLA-4-CD80/86, ICOS-B7RP1 and CD70-CD27 has been relatively well studied in SLE, recent data on the immunopathology of the CD137-CD137 ligand (CD137L) system in murine lupus models and patients with SLE highlight the critical role of this costimulatory system in initiating and perpetuating the diverse clinical and serological phenotypes of SLE. CD137, a membrane-bound receptor which belongs to the tumour necrosis factor receptor superfamily, is mainly expressed on activated T cells. Activation of the CD137 receptor via its interaction with CD137L which is expressed on antigen present cells (APC) including B cells, triggers bi-directional signalling; that is, signalling through CD137 as well as signalling through CD137L (reverse signalling), which further activates T cells and polarizes them to the Th1/Tc1 pathway. Further, via reverse CD137L signalling it enhances differentiation and maturation of the APC, particularly of dendritic cells, which subsequently drive proinflammatory cytokine production. In this review, recent data including our experience in the manipulation of CD137L signalling pertaining to the pathophysiology of SLE will be critically reviewed. More in-depth understanding of the biology of the CD137-CD137L co-stimulation system opens an opportunity to identify new prognostic biomarkers and the design of novel therapeutic approaches for advancing the management of SLE.

Activation of TGR5 with INT-777 attenuates oxidative stress and neuronal apoptosis via cAMP/PKCε/ALDH2 pathway after subarachnoid hemorrhage in rats

BackgroundOxidative stress and neuronal apoptosis play important roles in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). The activation of TGR5, a novel membrane-bound bile acid receptor, possesses anti-oxidative stress and anti-apoptotic effects in hepatobiliary disease and kidney disease. The present study aimed to explore the neuroprotective effect of TGR5 activation against EBI after SAH and the potential underlying mechanisms. MethodsThe endovascular perforation model of SAH was performed on 199 Sprague Dawley rats to investigate the beneficial effects of TGR5 activation after SAH. INT-777, a specific synthetic TGR5 agonist, was administered intranasally at 1 h after SAH induction. TGR5 CRISPR and ALDH2 CRISPR were administered intracerebroventricularly at 48 h before SAH to illuminate potential mechanisms. The SAH grade, short-term and long-term neurobehavioral tests, TUNEL staining, Fluoro-Jade C staining, Nissl staining, immunofluorescence staining, and western blots were performed at 24 h after SAH. ResultsThe expressions of endogenous TGR5 and ALDH2 gradually increased and peaked at 24 h after SAH. TGR5 was expressed primarily in neurons, as well as in astrocytes and microglia. The activation of TGR5 with INT-777 significantly improved the short-term and long-term neurological deficits, accompanied by reduced the oxidative stress and neuronal apoptosis at 24 h after SAH. Moreover, INT-777 treatment significantly increased the expressions of TGR5, cAMP, phosphorylated PKCε, ALDH2, HO-1, and Bcl-2, while downregulated the expressions of 4-HNE, Bax, and Cleaved Caspase-3. TGR5 CRISPR and ALDH2 CRISPR abolished the neuroprotective effects of TGR5 activation after SAH. ConclusionsIn summary, the activation of TGR5 with INT-777 attenuated oxidative stress and neuronal apoptosis via the cAMP/PKCε/ALDH2 signaling pathway after SAH in rats. Furthermore, TGR5 may serve as a novel therapeutic target to ameliorate EBI after SAH.

SpBark Suppresses Bacterial Infection by Mediating Hemocyte Phagocytosis in an Invertebrate Model, Scylla paramamosain.

Scavenger receptors are cell surface membrane-bound receptors that typically bind multiple ligands and promote the removal of endogenous proteins and pathogens. In this study, we characterized a novel scavenger receptor-like protein, namely, SpBark. SpBark was upregulated in hemocytes after challenges with bacteria, suggesting that it might be involved in antibacterial defense. SpBark is a type I transmembrane protein with four extracellular domains, including three scavenger receptor cysteine-rich domains (SRCRDs) and a C-type lectin domain (CTLD). Western blot assay showed that SpBark CTLD possessed a much stronger binding activity to tested microbes than the three SRCRDs. It also exhibited apparent binding activities to lipopolysaccharide (LPS) and acetylated low-density lipoprotein (ac-LDL), whereas the other SRCRDs showed much lower or no binding activities to these components. Agglutination activities were observed in the presence of Ca2+ by incubating microorganisms with SpBark CTLD instead of SRCRDs. These results suggested that SpBark CTLD was the major binding site for ac-LDL and LPS. Coating Vibrio parahemolyticus with SpBark CTLD promoted bacterial clearance in vivo. This finding indicated that SpBark might participate in the immune defenses against Gram-negative bacteria through a certain mechanism. The promotion of bacterial clearance by SpBark was further determined using SpBark-silenced crabs injected with V. parahemolyticus. SpBark knockdown by injection of SpBark dsRNA remarkably suppressed the clearance of bacteria in hemolymph. Meanwhile, it also severely restrained the phagocytosis of bacteria. This finding suggested that SpBark could modulate the phagocytosis of bacteria, and the promotion of bacterial clearance by SpBark was closely related to SpBark-mediated phagocytosis activity. The likely mechanism of bacterial clearance mediated by SpBark was as follows: SpBark acted as a pattern recognition receptor, which could sense and bind to LPS on the surface of invading bacteria with its CTLD in hemolymph. The binding to LPS made the bacteria adhere to the surface of hemocytes. This process would facilitate phagocytosis of the bacteria, resulting in their removal. This study provided new insights into the hemocyte phagocytosis mechanisms of invertebrates and the multiple biological functions of Bark proteins.

Understanding T cell signaling using membrane reconstitution.

T cells are central players of our immune system, as their functions range from killing tumorous and virus-infected cells to orchestrating the entire immune response. In order for T cells to divide and execute their functions, they must be activated by antigen-presenting cells (APCs) through a cell-cell junction. Extracellular interactions between receptors on T cells and their ligands on APCs trigger signaling cascades comprised of protein-protein interactions, enzymatic reactions, and spatial reorganization events, to either stimulate or repress T cell activation. Plasma membrane is the major platform for T cell signaling. Recruitment of cytosolic proteins to membrane-bound receptors is a common critical step in many signaling pathways. Membranes decrease the dimensionality of protein-protein interactions to enable weak yet biologically important interactions. Membrane resident proteins can phase separate into micro-islands that promote signaling by enriching or excluding signal regulators. Moreover, some membrane lipids can either mediate or regulate cell signaling by interacting with signaling proteins. While it is critical to investigate T cell signaling in a cellular environment, the large number of signaling pathways involved and potential crosstalk have made it difficult to obtain precise, quantitative information on T cell signaling. Reconstitution of purified proteins to modelmembranes provides a complementary avenue for T cell signaling research. Here, I review recent progress in studying T cell signaling using membrane reconstitution approaches.

Synergistic inhibition of GP130 and ERK signaling blocks chemoresistant bladder cancer cell growth

Multidrug resistance is a major treatment obstacle for recurrent and metastatic bladder cancer, which often leads to disease progression and poor clinical outcome. Although overexpression of interleukin-6 (IL-6) appears to play a critical role in the development of chemotherapy resistance, inhibitors for IL-6 alone have not improved clinical outcomes. Since the IL-6/IL-6R/GP130 complex is involved in multidrug resistance, another strategy would be to focus on glycoprotein-130 (GP130) since it dimerizes with IL-6R/CD26 as a membrane-bound signaling transducer receptor and initiates subsequent signaling activation and may be a potential therapeutic target. Currently, the role of GP130 in chemoresistant bladder cancer is unknown. In the present study, we demonstrate that GP130 is over-expressed in cisplatin and gemcitabine-resistant bladder cancer cells, and that the inhibition of GP130 expression significantly reduces cell viability, survival and migration. Downstream of GP130 is PI3K/AKT/mTOR signaling, which is inactivated by SC144, a GP130 inhibitor. However, Raf/MEK/ERK signaling, which also is downstream of GP130 is activated by SC144. This activation is likely based on a mTOR/S6K1/PI3K/ERK negative feedback loop, which is presumed to counteract the inhibitory effect of SC144 on tumor aggressiveness. Blocking both GP130 and pERK resulted in synergistic inhibition of cytotoxicity, clonal survival rates and cell migration in our chemotherapy resistant bladder cancer cells. This vertical inhibition offers a novel therapeutic strategy for targeting human chemoresistant bladder cancer.

Mechanism and Inhibition of Matrix Metalloproteinases.

Matrix metalloproteinases hydrolyze proteins and glycoproteins forming the extracellular matrix, cytokines and growth factors released in the extracellular space, and membrane-bound receptors on the outer cell membrane. The pathological relevance of MMPs has prompted the structural and functional characterization of these enzymes and the development of synthetic inhibitors as possible drug candidates. Recent studies have provided a better understanding of the substrate preference of the different members of the family, and structural data on the mechanism by which these enzymes hydrolyze the substrates. Here, we report the recent advancements in the understanding of the mechanism of collagenolysis and elastolysis, and we discuss the perspectives of new therapeutic strategies for targeting MMPs.

Mitofusin 2 Is Essential for IP3-Mediated SR/Mitochondria Metabolic Feedback in Ventricular Myocytes.

Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP3) by activating their membrane-bound receptors. We have previously demonstrated that IP3-mediated sarcoplasmic reticulum (SR) Ca2+ release results in mitochondrial Ca2+ uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP3-mediated SR/mitochondrial feedback in ventricular myocytes.Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca2+ uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates.Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca2+ uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca2+ uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca2+ uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca2+ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca2+ uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca2+ uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions.Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP3-mediated SR Ca2+ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication (IP3R-mRyR1 vs. ryanodine receptor type 2-mitochondrial calcium uniporter).

The Neonatal Fc Receptor (FcRn): A Misnomer?

Antibodies are essential components of an adaptive immune response. Immunoglobulin G (IgG) is the most common type of antibody found in circulation and extracellular fluids. Although IgG alone can directly protect the body from infection through the activities of its antigen binding region, the majority of IgG immune functions are mediated via proteins and receptors expressed by specialized cell subsets that bind to the fragment crystallizable (Fc) region of IgG. Fc gamma (γ) receptors (FcγR) belong to a broad family of proteins that presently include classical membrane-bound surface receptors as well as atypical intracellular receptors and cytoplasmic glycoproteins. Among the atypical FcγRs, the neonatal Fc receptor (FcRn) has increasingly gained notoriety given its intimate influence on IgG biology and its ability to also bind to albumin. FcRn functions as a recycling or transcytosis receptor that is responsible for maintaining IgG and albumin in the circulation, and bidirectionally transporting these two ligands across polarized cellular barriers. More recently, it has been appreciated that FcRn acts as an immune receptor by interacting with and facilitating antigen presentation of peptides derived from IgG immune complexes (IC). Here we review FcRn biology and focus on newer advances including how emerging FcRn-targeted therapies may affect the immune responses to IgG and IgG IC.

Heterogeneous Dielectric Implicit Membrane Model for the Calculation of MMPBSA Binding Free Energies.

Membrane-bound protein receptors are a primary biological drug target, but the computational analysis of membrane proteins has been limited. In order to improve molecular mechanics Poisson-Boltzmann surface area (MMPBSA) binding free energy calculations for membrane protein-ligand systems, we have optimized a new heterogeneous dielectric implicit membrane model, with respect to free energy simulations in explicit membrane and explicit water, and implemented it into the Amber software suite. This new model supersedes our previous uniform, single dielectric implicit membrane model by allowing the dielectric constant to vary with depth within the membrane. We calculated MMPBSA binding free energies for the human purinergic platelet receptor (P2Y12R) and two of the muscarinic acetylcholine receptors (M2R and M3R) bound to various antagonist ligands using both membrane models, and we found that the heterogeneous dielectric membrane model has a stronger correlation with experimental binding affinities compared to the older model under otherwise identical conditions. This improved membrane model increases the utility of MMPBSA calculations for the rational design and improvement of future drug candidates.

The interaction with fungal cell wall polysaccharides determines the salt tolerance of antifungal plant defensins

The fungal cell wall is the first point of contact between fungal pathogens and host organisms. It serves as a protective barrier against biotic and abiotic stresses and as a signal to the host that a fungal pathogen is present. The fungal cell wall is made predominantly of carbohydrates and glycoproteins, many of which serve as binding receptors for host defence molecules or activate host immune responses through interactions with membrane-bound receptors. Plant defensins are a large family of cationic antifungal peptides that protect plants against fungal disease. Binding of the plant defensin NaD1 to the fungal cell wall has been described but the specific component of the cell wall with which this interaction occurred was unknown. The effect of binding was also unclear, that is whether the plant defensin used fungal cell wall components as a recognition motif for the plant to identify potential pathogens or if the cell wall acted to protect the fungus against the defensin. Here we describe the interaction between the fungal cell wall polysaccharides chitin and β-glucan with NaD1 and other plant defensins. We discovered that the β-glucan layer protects the fungus against plant defensins and the loss of activity experienced by many cationic antifungal peptides at elevated salt concentrations is due to sequestration by fungal cell wall polysaccharides. This has limited the development of cationic antifungal peptides for the treatment of systemic fungal diseases in humans as the level of salt in serum is enough to inactivate most cationic peptides.

Response to IL-6 trans- and IL-6 classic signalling is determined by the ratio of the IL-6 receptor \u03b1 to gp130 expression: fusing experimental insights and dynamic modelling

BackgroundInterleukin-6 is a pleiotropic cytokine with high clinical relevance and an important mediator of cellular communication, orchestrating both pro- and anti-inflammatory processes. Interleukin-6-induced signalling is initiated by binding of IL-6 to the IL-6 receptor α and subsequent binding to the signal transducing receptor subunit gp130. This active receptor complex initiates signalling through the Janus kinase/signal transducer and activator of transcription pathway. Of note, IL-6 receptor α exists in a soluble and a transmembrane form. Binding of IL-6 to membrane-bound IL-6 receptor α induces anti-inflammatory classic signalling, whereas binding of IL-6 to soluble IL-6 receptor α induces pro-inflammatory trans-signalling. Trans-signalling has been described to be markedly stronger than classic signalling. Understanding the molecular mechanisms that drive differences between trans- and classic signalling is important for the design of trans-signalling-specific therapies. These differences will be addressed here using a combination of dynamic mathematical modelling and molecular biology.MethodsWe apply an iterative systems biology approach using set-based modelling and validation approaches combined with quantitative biochemical and cell biological analyses.ResultsThe combination of experimental analyses and dynamic modelling allows to relate the observed differences between IL-6-induced trans- and classic signalling to cell-type specific differences in the expression and ratios of the individual subunits of the IL-6 receptor complex. Canonical intracellular Jak/STAT signalling is indifferent in IL-6-induced trans- and classic signalling.ConclusionThis study contributes to the understanding of molecular mechanisms of IL-6 signal transduction and underlines the power of combined dynamical modelling, model-based validation and biological experiments. The opposing pro- and anti-inflammatory responses initiated by IL-6 trans- and classic signalling depend solely on the expression ratios of the subunits of the entire receptor complex. By pointing out the importance of the receptor expression ratio for the strength of IL-6 signalling this study lays a foundation for future precision medicine approaches that aim to selectively block pro-inflammatory trans-signalling. Furthermore, the derived models can be used for future therapy design.Graphical abstract