Ligand Signaling Research Articles

Abstract 4786: Imipridones induce lipid peroxidation and oxidative stress in gastrointestinal malignancies

Abstract Colorectal and pancreatic cancer collectively comprise 20% of all cancer-related deaths in the United States. Despite therapeutic advancements, the current standard of care often results in significant toxicity. Therefore, there is an urgent need to develop more efficacious novel therapies. The imipridones, ONC201/TIC10, ONC206, and ONC212, are novel small-molecules that bind mitochondrial ClpP and induce activation of the integrated stress response (ISR), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling, and oxidative stress. Each has previously demonstrated antitumor effects in a variety of preclinical in vitro and in vivo cancer subtypes, including colorectal cancer, with limited toxicity. Reactive oxidation species (ROS) are natural biproducts of cellular oxidative metabolism and play important roles in the modulation of cell death pathways, differentiation, and immune response. Using varying doses of ONC201, ONC206, and ONC212, we observed increased lipid peroxidation in several colorectal and pancreatic cancer cell lines using the C-11 BODIPY assay which measures lipid-specific peroxides. Lipid peroxidation was reversed by the ferroptosis inhibitor liproxstatin-1, suggesting that in addition to inducing apoptosis, imipridones may be involved in ferroptosis induction. Future directions include mechanistic work, investigation of the effect of imipridone-induced lipid peroxidation on immune cell cytotoxicity, and understanding the relative importance of lipid peroxidation in the clinical efficacy of imipridones. Citation Format: Arielle De La Cruz, Praveen R. Srinivasan, Andrew George, Varun V. Prabhu, Maximilian P. Pinho-Schwermann, Wafik S. El-Deiry. Imipridones induce lipid peroxidation and oxidative stress in gastrointestinal malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4786.

Abstract 6382: Activation of transcription factors, p65 and NFATc1, mediated by WNT5a, induces up-regulation of novel immune checkpoint CNTN4 expression

Abstract We previously confirmed that contactin 4 (CNTN4) is highly expressed in the various human tumor tissues and inhibits mouse and human T cell activity by suppressing a cascade of signaling processes associated with T cell activation through binding with amyloid precursor protein (APP) of T cells. However, the molecular mechanism for the expression of CNTN4 in tumors has yet to be elucidated. So, we investigated whether oncogenic ligands regulate the expression of CNTN4 through CNTN4 promoter (- 2,000 bp ~ + 100 bp)-based reporter gene assays in HEK293 cells using oncogenic ligands. Here, we identified that WNT5a significantly increased the expression of CNTN4 in a dose-dependent manner. Furthermore, the treatment of the WNT5a ligand in the human colorectal cancer cell line SW480, which expresses CNTN4 at a low level, increased the expression of CNTN4. In addition, CNTN4 expression was decreased when the WNT5a antagonist (Box5, a WNT5a-derived N-butyloxycarbonyl hexapeptide) was treated in the human osteosarcoma cell line U2OS expressing CNTN4 endogenously. The changes in the expression of CNTN4 were observed by RT-PCR and FACS. Treatment of the WNT5a ligand or WNT5a antagonist changed the transcriptional expression of CNTN4 in each cell line and affected the cell surface expression of CNTN4. Since WNT5a ultimately increases the activity of transcription factors such as NFATc1 or p65 to increase the transcriptional expression of the target gene, we further investigated the effect of NFATc1 or p65 on the expression of CNTN4. We found that NFATc1 or p65 binds to the promoter of CNTN4 and increases the expression of transcription. Moreover, it was recently reported that anti-PD-1-refractory melanoma exhibits elevated WNT ligand signaling activity, especially upregulated WNT7b and WNT5a in the WNT ligands. Following this background, the expression level of CNTN4 can be increased in the refractory environment to immunotherapy induced by elevated WNT signaling. Therefore, we constructed B16F10 murine melanoma cells overexpressing WNT5a (B16F10/WNT5a) to assess the anti-tumor efficacy by administering GENA-104A16, the anti-CNTN4 humanized monoclonal antibody, in the syngeneic mice model with increased expression of CNTN4 induced by WNT5a, and in vivo efficacy by treatment of GENA-104A16 is under assessment in the B16F10/WNT5a syngeneic mice model. Through the investigation, we expect to support the rationale and strategy for a combination therapy approach of GENA-104A16 for non-responding patients to immunotherapy. Citation Format: Mi Young Cha, Bu-Nam Jeon, Hyeokgu Kang, Kyung Mi Park, Hansoo Park. Activation of transcription factors, p65 and NFATc1, mediated by WNT5a, induces up-regulation of novel immune checkpoint CNTN4 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6382.

Computational modeling of TGF-β2:TβRI:TβRII receptor complex assembly as mediated by the TGF-β coreceptor betaglycan

Transforming growth factor-β1, -β2, and -β3 (TGF-β1, -β2, and -β3) are secreted signaling ligands that play essential roles in tissue development, tissue maintenance, immune response, and wound healing. TGF-β ligands form homodimers and signal by assembling a heterotetrameric receptor complex comprised of two type I receptor (TβRI):type II receptor (TβRII) pairs. TGF-β1 and TGF-β3 ligands signal with high potency due to their high affinity for TβRII, which engenders high-affinity binding of TβRI through a composite TGF-β:TβRII binding interface. However, TGF-β2 binds TβRII 200–500 more weakly than TGF-β1 and TGF-β3 and signals with lower potency compared with these ligands. Remarkably, the presence of an additional membrane-bound coreceptor, known as betaglycan, increases TGF-β2 signaling potency to levels similar to TGF-β1 and -β3. The mediating effect of betaglycan occurs even though it is displaced from and not present in the heterotetrameric receptor complex through which TGF-β2 signals. Published biophysics studies have experimentally established the kinetic rates of the individual ligand-receptor and receptor-receptor interactions that initiate heterotetrameric receptor complex assembly and signaling in the TGF-β system; however, current experimental approaches are not able to directly measure kinetic rates for the intermediate and latter steps of assembly. To characterize these steps in the TGF-β system and determine the mechanism of betaglycan in the potentiation of TGF-β2 signaling, we developed deterministic computational models with different modes of betaglycan binding and varying cooperativity between receptor subtypes. The models identified conditions for selective enhancement of TGF-β2 signaling. The models provide support for additional receptor binding cooperativity that has been hypothesized but not evaluated in the literature. The models further showed that betaglycan binding to the TGF-β2 ligand through two domains provides an effective mechanism for transfer to the signaling receptors that has been tuned to efficiently promote assembly of the TGF-β2(TβRII)2(TβRI)2 signaling complex.

A c-di-GMP binding effector controls cell size in a cyanobacterium

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule. It is also a critical player in the regulation of cell size and cell behaviors such as cell aggregation and phototaxis in cyanobacteria, which constitute an important group of prokaryotes for their roles in the ecology and evolution of the Earth. However, c-di-GMP receptors have never been revealed in cyanobacteria. Here, we report the identification of a c-di-GMP receptor, CdgR, from the filamentous cyanobacterium Anabaena PCC 7120. Crystal structural analysis and genetic studies demonstrate that CdgR binds c-di-GMP at the dimer interface and this binding is required for the control of cell size in a c-di-GMP-dependent manner. Different functions of CdgR, in ligand binding and signal transmission, could be separated genetically, allowing us to dissect its molecular signaling functions. The presence of the apo-form of CdgR triggers cell size reduction, consistent with the similar effects observed with a decrease of c-di-GMP levels in cells. Furthermore, we found that CdgR exerts its function by interacting with a global transcription factor DevH, and this interaction was inhibited by c-di-GMP. The lethal effect triggered by conditional depletion of DevH or by the production of several point-mutant proteins of CdgR in cells indicates that this signaling pathway plays critical functions in Anabaena. Our studies revealed a mechanism of c-di-GMP signaling in the control of cell size, an important and complex trait for bacteria. CdgR is highly conserved in cyanobacteria, which will greatly expand our understanding of the roles of c-di-GMP signaling in these organisms.

Potential Impact of Polymorphisms in Toll-like Receptors 2, 3, 4, 7, 9, miR-146a, miR-155, and miR-196a Genes on Osteoarthritis Susceptibility.

Osteoarthritis (OA) is a progressive inflammatory disease of synovial joints and a leading cause of disability among adults. Inflammation-related genes, including genes for Toll-like receptors (TLRs), are tightly controlled by several microRNAs that, in addition to their pivotal role in the epigenetic regulation of target genes, are ligands for TLR activation and downstream signaling. Thus, we evaluated the association between OA risk and genetic variants in TLR2, TLR3, TLR4, TLR7, TLR9, and microRNAs that regulate TLRs signaling miR146a, miR155, and miR196a2. Our study group consisted of 95 surgically treated OA patients and a control group of 104 healthy individuals. Genetic polymorphisms were determined using TaqMan real-time PCR assays (Applied Biosystems). Adjusted logistic regression analysis demonstrated that polymorphisms in TLR4 rs4986790 (OR = 2.964, p = 0.006), TLR4 rs4986791 (OR = 8.766, p = 0.00001), and TLR7 rs385389 (OR = 1.579, p = 0.012) increased OA risk, while miR-196a2 rs11614913 (OR = 0.619, p = 0.034) was significantly associated with decreased OA risk. Our findings indicate that polymorphisms in the TLR4 and TLR7 genes might increase OA risk and suggest a novel association of miR-196a2 polymorphism with decreased OA susceptibility. The modulation of TLRs and miRNAs and their cross-talk might be an attractive target for a personalized approach to OA management.

Lack of evidence that fibrillin1 regulates bone morphogenetic protein 4 activity in kidney or lung.

The Bone morphogenetic protein 4 (BMP4) precursor protein is cleaved at two sites to generate an active ligand and inactive prodomain. The ligand and prodomain form a noncovalent complex following the first cleavage, but dissociate after the second cleavage. Transient formation of this complex is essential to generate a stable ligand. Fibrillins (FBNs) bind to the prodomains of BMPs, and can regulate the activity of some ligands. Whether FBNs regulate BMP4 activity is unknown. Mice heterozygous for a null allele of Bmp4 showed incompletely penetrant kidney defects and females showed increased mortality between postnatal day 6 and 8. Removal of one copy of Fbn1 did not rescue or enhance kidney defects or lethality. The lungs of Fbn1+/- females had enlarged airspaces that were unchanged in Bmp4+/- ;Fbn1+/- mice. Additionally, removal of one or both alleles of Fbn1 had no effect on steady state levels of BMP4 ligand or on BMP activity in postnatal lungs. These findings do not support the hypothesis that FBN1 plays a role in promoting BMP4 ligand stability or signaling, nor do they support the alternative hypothesis that FBN1 sequesters BMP4 in a latent form, as is the case for other BMP family members.

Novel scFv against Notch Ligand JAG1 Suitable for Development of Cell Therapies toward JAG1-Positive Tumors

The Notch signaling ligand JAG1 is overexpressed in various aggressive tumors and is associated with poor clinical prognosis. Hence, therapies targeting oncogenic JAG1 hold great potential for the treatment of certain tumors. Here, we report the identification of specific anti-JAG1 single-chain variable fragments (scFvs), one of them endowing chimeric antigen receptor (CAR) T cells with cytotoxicity against JAG1-positive cells. Anti-JAG1 scFvs were identified from human phage display libraries, reformatted into full-length monoclonal antibodies (Abs), and produced in mammalian cells. The characterization of these Abs identified two specific anti-JAG1 Abs (J1.B5 and J1.F1) with nanomolar affinities. Cloning the respective scFv sequences in our second- and third-generation CAR backbones resulted in six anti-JAG1 CAR constructs, which were screened for JAG1-mediated T-cell activation in Jurkat T cells in coculture assays with JAG1-positive cell lines. Studies in primary T cells demonstrated that one CAR harboring the J1.B5 scFv significantly induced effective T-cell activation in the presence of JAG1-positive, but not in JAG1-knockout, cancer cells, and enabled specific killing of JAG1-positive cells. Thus, this new anti-JAG1 scFv represents a promising candidate for the development of cell therapies against JAG1-positive tumors.

Genomic, epigenomic, and transcriptomic signatures of prostate cancer between African American and European American patients.

Prostate cancer is the second most common cancer in men in the United States, and racial disparities are greatly observed in the disease. Specifically, African American (AA) patients have 60% higher incidence and mortality rates, in addition to higher grade and stage prostate tumors, than European American (EA) patients. In order to narrow the gap between clinical outcomes for these two populations, genetic and molecular signatures contributing to this disparity have been characterized. Over the past decade, profiles of prostate tumor samples from different ethnic groups have been developed using molecular and functional assays coupled with next generation sequencing or microarrays. Comparative genome-wide analyses of genomic, epigenomic, and transcriptomic profiles from prostate tumor samples have uncovered potential race-specific mutations, copy number alterations, DNA methylation, and gene expression patterns. In this study, we reviewed over 20 published studies that examined the aforementioned molecular contributions to racial disparities in AA and EA prostate cancer patients. The reviewed genomic studies revealed mutations, deletions, amplifications, duplications, or fusion genes differentially enriched in AA patients relative to EA patients. Commonly reported genomic alterations included mutations or copy number alterations of FOXA1, KMT2D, SPOP, MYC, PTEN, TP53, ZFHX3, and the TMPRSS2-ERG fusion. The reviewed epigenomic studies identified that CpG sites near the promoters of PMEPA1, RARB, SNRPN, and TIMP3 genes were differentially methylated between AA and EA patients. Lastly, the reviewed transcriptomic studies identified genes (e.g. CCL4, CHRM3, CRYBB2, CXCR4, GALR1, GSTM3, SPINK1) and signaling pathways dysregulated between AA and EA patients. The most frequently found dysregulated pathways were involved in immune and inflammatory responses and neuroactive ligand signaling. Overall, we observed that the genomic, epigenomic, and transcriptomic alterations evaluated between AA and EA prostate cancer patients varied between studies, highlighting the impact of using different methods and sample sizes. The reported genomic, epigenomic, and transcriptomic alterations do not only uncover molecular mechanisms of tumorigenesis but also provide researchers and clinicians valuable resources to identify novel biomarkers and treatment modalities to improve the disparity of clinical outcomes between AA and EA patients.

Sotatercept analog improves cardiopulmonary remodeling and pulmonary hypertension in experimental left heart failure.

Pulmonary hypertension due to left heart disease (PH-LHD) is the most frequent manifestation of PH but lacks any approved treatment. Activin receptor type IIA-Fc fusion protein (ActRIIA-Fc) was found previously to be efficacious in experimental and human pulmonary arterial hypertension (PAH). Here we tested the hypothesis that ActRIIA-Fc improves pulmonary vascular remodeling and alleviates PH in models of PH-LHD, specifically in subtypes of heart failure with reduced ejection fraction (PH-HFrEF) and preserved ejection fraction (PH-HFpEF). Treatment with murine ActRIIA-Fc reduced cardiac remodeling and improved cardiac function in two mouse models of left heart disease without PH, confirming that this inhibitor of activin-class ligand signaling can exert cardioprotective effects in heart failure. In a mouse model of PH-HFrEF with prolonged pressure overload caused by transverse aortic constriction, ActRIIA-Fc treatment significantly reduced pulmonary vascular remodeling, pulmonary fibrosis, and pulmonary hypertension while exerting beneficial structural, functional, and histological effects on both the left and right heart. Additionally, in an obese ZSF1-SU5416 rat model of PH-HFpEF with metabolic dysregulation, therapeutic treatment with ActRIIA-Fc normalized SMAD3 overactivation in pulmonary vascular and perivascular cells, reversed pathologic pulmonary vascular and cardiac remodeling, improved pulmonary and cardiac fibrosis, alleviated PH, and produced marked functional improvements in both cardiac ventricles. Studies in vitro revealed that treatment with ActRIIA-Fc prevents an abnormal, glucose-induced, activin-mediated, migratory phenotype in human pulmonary artery smooth muscle cells, providing a mechanism by which ActRIIA-Fc could exert therapeutic effects in experimental PH-HFpEF with metabolic dysregulation. Our results demonstrate that ActRIIA-Fc broadly corrects cardiopulmonary structure and function in experimental PH-LHD, including models of PH-HFrEF and PH-HFpEF, leading to alleviation of PH under diverse pathophysiological conditions. These findings highlight the important pathogenic contributions of activin-class ligands in multiple forms of experimental PH and support ongoing clinical evaluation of human ActRIIA-Fc (sotatercept) in patients with PH-HFpEF.

Surgical Management and Denosumab for Aneurysmal Bone Cysts of the Spine in an Australian Tertiary Paediatric Centre.

Aneurysmal bone cysts (ABC) are rare osteolytic, benign but often locally aggressive tumours of the long bones or vertebrae. For spinal ABC, surgical management, embolisation or sclerotherapy alone often carry high morbidity and/or high recurrence rates. Interruption of receptor activator of nuclear factor-kappa B ligand (RANKL) signalling holds promise as an effective therapeutic strategy for these tumours. We aimed to review the approach to surgical management and evaluate the efficacy and safety of denosumab for ABC of the spine in children. Retrospective review of 7 patients treated with denosumab using a standardised protocol for ABC of the spine in a tertiary paediatric centre. Surgical intervention was only conducted if there was spinal instability or significant neurological impairment. Denosumab 70mg/m2 was given 4-weekly for at least 6months, followed by 2 doses of zoledronate 0.025mg/kg, aiming to prevent rebound hypercalcaemia. All patients achieved stability of the spine and resolution of neurological impairment, if present. Six patients achieved metabolic remission and have ceased denosumab without recurrence to date; the other showed clinical and radiological improvement without complete metabolic remission. Three patients developed symptomatic hypercalcaemia 5-7months after cessation of denosumab, requiring additional bisphosphonate treatment. We present our algorithm for the surgical and medical management of paediatric spinal ABC. Denosumab produced a radiological and metabolic response in all patients, with complete remission in most. Follow-up time was not long enough to evaluate the endurance of response after cessation in some patients. Incidence of rebound hypercalcaemia in this paediatric cohort was high, prompting a change to our protocol.

Prospect of acromegaly therapy: molecular mechanism of clinical drugs octreotide and paltusotine

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors and represents as a therapeutic target. Several peptide analogs mimicking the endogenous ligand somatostatin are available for clinical use, but poor therapeutic effects occur in a subset of patients, which may be correlated with subtype selectivity or cell surface expression. Here, we clarify the signal bias profiles of the first-generation peptide drug octreotide and a new-generation small molecule paltusotine by evaluating their pharmacological characteristics. We then perform cryo-electron microscopy analysis of SSTR2-Gi complexes to determine how the drugs activate SSTR2 in a selective manner. In this work, we decipher the mechanism of ligand recognition, subtype selectivity and signal bias property of SSTR2 sensing octreotide and paltusotine, which may aid in designing therapeutic drugs with specific pharmacological profiles against neuroendocrine tumors.

Interleukin-27-dependent transcriptome signatures during neonatal sepsis.

Human newborns exhibit increased vulnerability and risk of mortality from infection that is consistent with key differences in the innate and adaptive immune responses relative to those in adult cells. We have previously shown an increase in the immune suppressive cytokine, IL-27, in neonatal cells and tissues from mice and humans. In a murine model of neonatal sepsis, mice deficient in IL-27 signaling exhibit reduced mortality, increased weight gain, and better control of bacteria with reduced systemic inflammation. To explore a reprogramming of the host response in the absence of IL-27 signaling, we profiled the transcriptome of the neonatal spleen during Escherichia coli-induced sepsis in wild-type (WT) and IL-27Rα-deficient (KO) mice. We identified 634 genes that were differentially expressed, and those most upregulated in WT mice were associated with inflammation, cytokine signaling, and G protein coupled receptor ligand binding and signaling. These genes failed to increase in the IL-27Rα KO mice. We further isolated an innate myeloid population enriched in macrophages from the spleens of control and infected WT neonates and observed similar changes in gene expression aligned with changes in chromatin accessibility. This supports macrophages as an innate myeloid population contributing to the inflammatory profile in septic WT pups. Collectively, our findings highlight the first report of improved pathogen clearance amidst a less inflammatory environment in IL-27Rα KO. This suggests a direct relationship between IL-27 signaling and bacterial killing. An improved response to infection that is not reliant upon heightened levels of inflammation offers new promise to the potential of antagonizing IL-27 as a host-directed therapy for neonates.

Exploration of the heterogeneity and interaction of epithelial cells and NK/T-cells in Laryngeal Squamous Cell Carcinoma based on single-cell RNA sequencing data

ObjectivesWe aimed to explore the heterogeneity and differentiation trajectories of epithelial cells and NK/T-cells in Laryngeal Squamous Cell Carcinoma (LSCC). MethodsWe downloaded the GSE150321 data set containing LSCC01 and LSCC02 samples single cell RNA data from Gene Expression Omnibus. The UMAP analysis was performed to identify the cell subpopulations and cell locations of subpopulations. Seurat package was used to analyze the differential expression of genes. The function of differential expression genes was analyzed using DAVID database. The monocle2 package was used to analyze differentiation trajectories. We used the CellChat package to observe the signaling pathways and ligand–receptor pairs for epithelial cells and NK/T-cells. ResultsAll the LSCC cells were divided into 16 subpopulation that included 7 epithelial cell subsets, 3 T-cell subsets. The function analysis indicated that epithelial cells and NK/T-cells mainly participated in different process, such as cell cycle, immune response, and cell migration. Then, the results of differentiation trajectory indicated that the ability of migration, and the activation of the immune system increases, while the ability of apoptosis, and glucose metabolic process decreases as pseudotime. Migration-related epithelial cells act on all T-cells via the CNTN2-CNTN2 ligand–receptor pair, which suggested that CNTN2 might be an important biomarker for regulating migration of epithelial cells. ConclusionsOur study characterized the heterogeneity of LSCC, which provided novel insights into LSCC and identified a new mechanism and target for clinical LSCC threapies. EvidenceIV.

Therapeutic potential of allosteric modulators for the treatment of gastrointestinal motility disorders.

Gastrointestinal motility is tightly regulated by the enteric nervous system (ENS). Disruption of coordinated enteric nervous system activity can result in dysmotility. Pharmacological treatment options for dysmotility include targeting of G protein-coupled receptors (GPCRs) expressed by neurons of the enteric nervous system. Current GPCR-targeting drugs for motility disorders bind to the highly conserved endogenous ligand-binding site and promote indiscriminate activation or inhibition of the target receptor throughout the body. This can be associated with significant side-effect liability and a loss of physiological tone. Allosteric modulators of GPCRs bind to a distinct site from the endogenous ligand, which is typically less conserved across multiple receptor subtypes and can modulate endogenous ligand signalling. Allosteric modulation of GPCRs that are important for enteric nervous system function may provide effective relief from motility disorders while limiting side-effects. This review will focus on how allosteric modulators of GPCRs may influence gastrointestinal motility, using 5-hydroxytryptamine (5-HT), acetylcholine (ACh) and opioid receptors as examples. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.

Evolutionary features of ligands and their receptors via protein-protein interactions and essentiality in primates.

Protein evolution rate is negatively correlated with several effectors, such as expression level, expression distribution, protein-protein interactions (PPIs), and essentiality for survival. These effectors can characterize the signaling pathways mediated by ligand-receptor binding. However, it is unclear whether these effectors are constraining factors on the pathway-specific evolution of ligands and receptors. To clarify the relation between the effectors and protein evolution (dN /dS ratio) in ligands and their receptors considering each signaling pathway, we investigated 377 proteins in 20 peptide/protein ligand groups and their receptor groups using 15 primate sequences. The dN /dS ratios between peptide/protein ligand groups and their receptor groups were positively correlated, suggesting the protein evolution under the influence of signaling pathway to which they belong. Comparing each signaling pathway, ligands and receptors mainly related to development and growth (FGF/Hedgehog/Notch/WNT groups) showed lower dN /dS ratios, higher PPI numbers, and higher essentiality, whereas those mainly related to immune process (CSF/IFN/IL/TNF groups) showed higher dN /dS ratios, lower PPI numbers, and lower essentiality. Most ligands and receptors were poorly expressed, and expression level was not a constraining factor on the protein evolution. These findings indicate that PPI and essentiality are constraining factors that characterize the pathway-specific evolution of ligands and receptors.

Impact of membrane lipid polyunsaturation on dopamine D2 receptor ligand binding and signaling.

Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of β-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

Chondroitin sulfate glycosaminoglycans function as extra/pericellular ligands for cell surface receptors.

Chondroitin sulfate (CS) chains, a class of sulfated glycosaminoglycan (GAG) polysaccharides, are ubiquitously distributed in extra/pericellular matrices that establish microenvironmental niches to support a multitude of cellular events. Such wide-ranging functions of CS chains are attributable not only to their sulfation pattern-dependent structural divergence, but also to their multiple modes of action. Although it has long been accepted that CS chains act as passive structural scaffolds that often behave as co-receptors and/or reservoirs for various humoral factors, the discovery of cell surface receptor molecules for distinct CS chains has offered insights into a novel mode of CS function as dynamic extra/pericellular signaling ligands. A recent report by Gong et al. (Identification of PTPRσ-interacting proteins by proximity-labeling assay. J. Biochem. 2021; 169:187-194) also strongly reinforced the physiological importance of CS receptor-mediated signaling pathways. In this commentary, we briefly introduce the functional aspects of CS chains as extra/pericellular signaling molecules.

The role played by ailanthone in inhibiting bone metastasis of breast cancer by regulating tumor-bone microenvironment through the RANKL-dependent pathway.

Introduction: Bone metastasis of breast cancer (BC) is a process in which the disruption of the bone homeostatic microenvironment leads to an increase in osteoclast differentiation. Ailanthus altissima shows an inhibitory effect on osteoclast differentiation. Ailanthone (AIL) refers to a natural compound isolated from Ailanthus altissima, a Chinese herbal medicine, and has effective anti-tumor activity in numerous cell lines. Its impact on bone metastases for BC is yet unclear. Methods: We measured the effect of AIL on MDA-MB-231 cells by wound healing experiments, Transwell and colony formation experiment. Using the Tartrate-resistant Acid Phosphatase (TRAP) staining tests, filamentous (F-actin) staining and bone resorption test to detect the effect of AIL on the osteoclast cell differentiation of the Bone Marrow-derived Macrophages (BMMs), activated by the MDA-MB-231 cell Conditioned Medium (MDA-MB-231 CM) and the Receptor Activator of Nuclear factor-κB Ligand (RANKL),and to explore its possibility Mechanisms. In vivo experiments verified the effect of AIL on bone destruction in breast cancer bone metastasis model mice. Results: In vitro, AIL significantly decrease the proliferation, migration and infiltration abilities of MDA-MB-231 cells at a safe concentration, and also reduced the expression of genes and proteins involved in osteoclast formation in MDA-MB-231 cells. Osteoclast cell differentiation of the BMMs, activated by MDA-MB-231 CM and RANKL, were suppressed by AIL in the concentration-dependent manner. Additionally, it inhibits osteoclast-specific gene and protein expression. It was noted that AIL inhibited the expression of the osteoclast differentiation-related cytokines RANKL and interleukin-1β (IL-1β) that were secreted by the MDA-MB-231 cells after upregulating the Forkhead box protein 3 (FOXP3) expression. Furthermore, AIL also inhibits the expression of the Mitogen-Activated Protein Kinase (MAPK), Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), and Nuclear factor-κB Ligand (NF-κB) signaling pathways, which then suppresses the MDA-MB-231CM-induced development of Osteoclasts. Conclusion: Our study shows that AIL blocks osteoclast differentiation in the bone metastasis microenvironment by inhibiting cytokines secreted by BC cells, which may be a potential agent for the treatment of BC and its secondary bone metastasis.

Unique ligand and kinase-independent roles of the insulin receptor in regulation of cell cycle, senescence and apoptosis

Insulin acts through the insulin receptor (IR) tyrosine kinase to exert its classical metabolic and mitogenic actions. Here, using receptors with either short or long deletion of the β-subunit or mutation of the kinase active site (K1030R), we have uncovered a second, previously unrecognized IR signaling pathway that is intracellular domain-dependent, but ligand and tyrosine kinase-independent (LYK-I). These LYK-I actions of the IR are linked to changes in phosphorylation of a network of proteins involved in the regulation of extracellular matrix organization, cell cycle, ATM signaling and cellular senescence; and result in upregulation of expression of multiple extracellular matrix-related genes and proteins, down-regulation of immune/interferon-related genes and proteins, and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent (LYK-D) signaling, the IR regulates a second, ligand and tyrosine kinase-independent (LYK-I) pathway, which regulates the cellular machinery involved in senescence, matrix interaction and response to extrinsic challenges.

The effect of ACVR1B/TGFBR1/ACVR1C signaling inhibition on oocyte and granulosa cell development during in vitro growth culture.

The signals of the transforming growth factor β (TGF-β) superfamily play a critical role in follicular development in mammals. ACVR1B/TGFBR1/ACVR1C receptors mediate the signaling of several TGF-β superfamily ligands in granulosa cells. Although the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in follicular development has been confirmed using mutant mouse models, the detailed roles of the signaling in granulosa cell and oocyte development have not been clearly defined. In the present study, we examined the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in granulosa cells using an in vitro growth culture of oocyte-granulosa cell complexes (OGCs) and SB431542, a potent inhibitor of the receptor signaling. Although cumulus-oocyte complexes isolated from the control OGCs were able to undergo cumulus expansion, those isolated from OGCs grown with the inhibitor were not competent, even in the presence of in vivo-grown oocytes. The diameter of the oocytes in the SB431542-treated OGCs was comparable with that of the control; however, these oocytes were not competent for complete meiotic maturation or preimplantation development. Therefore, ACVR1B/TGFBR1/ACVR1C receptor signaling is not required for oocytes to increase their volume but is essential for the normal development of cumulus cells and oocyte developmental competence.