Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

Ligand recognition and G-protein coupling of trace amine receptor TAAR1.

Trace-amine-associated receptors (TAARs), a group of biogenic amine receptors, have essential roles in neurological and metabolic homeostasis1. They recognize diverse endogenous trace amines and subsequently activate a range of G-protein-subtype signalling pathways2,3. Notably, TAAR1 has emerged as a promising therapeutic target for treating psychiatric disorders4,5. However, the molecular mechanisms underlying its ability to recognize different ligands remain largely unclear. Here we present nine cryo-electron microscopy structures, with eight showing human and mouse TAAR1 in a complex with an array of ligands, including the endogenous 3-iodothyronamine, two antipsychotic agents, the psychoactive drug amphetamine and two identified catecholamine agonists, and one showing 5-HT1AR in a complex with an antipsychotic agent. These structures reveal a rigid consensus binding motif in TAAR1 that binds to endogenous trace amine stimuli and two extended binding pockets that accommodate diverse chemotypes. Combined with mutational analysis, functional assays and molecular dynamic simulations, we elucidate the structural basis of drug polypharmacology and identify the species-specific differences between human and mouse TAAR1. Our study provides insights into the mechanism of ligand recognition and G-protein selectivity by TAAR1, which may help in the discovery of ligands or therapeutic strategies for neurological and metabolic disorders.

Substance P promotes epidural fibrosis via induction of type 2 macrophages.

In response to spinal surgery, neurons secrete a large amount of substance P into the epidural area. Substance P is involved in macrophage differentiation and fibrotic disease. However, the specific roles and mechanisms of substance P in epidural fibrosis remain unclear. In this study, we established a mouse model of L1-L3 laminectomy and found that dorsal root ganglion neurons and the macrophages infiltrating into the wound area released sphingolipids. In vitro experiments revealed that type 1 macrophages secreted substance P, which promoted differentiation of type 1 macrophages towards a type 2 phenotype. High-throughput mRNA-seq analysis revealed that the sphingolipid metabolic pathway may be involved in the regulation of type 2 macrophages by substance P. Specifically, sphingomyelin synthase 2, a component of the sphingolipid metabolic pathway, promoted M2 differentiation in substance P-treated macrophages, while treating the macrophages with LY93, a sphingomyelin synthase 2 inhibitor, suppressed M2 differentiation. In addition, substance P promoted the formation of neutrophil extracellular traps, which further boosted M2 differentiation. Blocking substance P with the neurokinin receptor 1 inhibitor RP67580 decreased the number of M2 macrophages in the wound area after spinal surgery and alleviated epidural fibrosis, as evidenced by decreased fibronectin, α-smooth muscle actin, and collagen I in the scar tissue. These results demonstrated that substance P promotes M2 macrophage differentiation in epidural fibrosis via sphingomyelin synthase 2 and neutrophil extracellular traps. These findings provide a novel strategy for the treatment of epidural fibrosis.

Structural basis and molecular mechanism of biased GPBAR signaling in regulating NSCLC cell growth via YAP activity.

The G protein-coupled bile acid receptor (GPBAR) is the membrane receptor for bile acids and a driving force of the liver-bile acid-microbiota-organ axis to regulate metabolism and other pathophysiological processes. Although GPBAR is an important therapeutic target for a spectrum of metabolic and neurodegenerative diseases, its activation has also been found to be linked to carcinogenesis, leading to potential side effects. Here, via functional screening, we found that two specific GPBAR agonists, R399 and INT-777, demonstrated strikingly different regulatory effects on the growth and apoptosis of non-small cell lung cancer (NSCLC) cells both in vitro and in vivo. Further mechanistic investigation showed that R399-induced GPBAR activation displayed an obvious bias for ß-arrestin 1 signaling, thus promoting YAP signaling activation to stimulate cell proliferation. Conversely, INT-777 preferentially activated GPBAR-Gs signaling, thus inactivating YAP to inhibit cell proliferation and induce apoptosis. Phosphorylation of GPBAR by GRK2 at S310/S321/S323/S324 sites contributed to R399-induced GPBAR-ß-arrestin 1 association. The cryoelectron microscopy (cryo-EM) structure of the R399-bound GPBAR-Gs complex enabled us to identify key interaction residues and pivotal conformational changes in GPBAR responsible for the arrestin signaling bias and cancer cell proliferation. In summary, we demonstrate that different agonists can regulate distinct functions of cell growth and apoptosis through biased GPBAR signaling and control of YAP activity in a NSCLC cell model. The delineated mechanism and structural basis may facilitate the rational design of GPBAR-targeting drugs with both metabolic and anticancer benefits.

Progesterone activates GPR126 to promote breast cancer development via the Gi pathway.

GPR126 is a member of the adhesion G protein-coupled receptors (aGPCRs) that is essential for the normal development of diverse tissues, and its mutations are implicated in various pathological processes. Here, through screening 34 steroid hormones and their derivatives for cAMP production, we found that progesterone (P4) and 17-hydroxyprogesterone (17OHP) could specifically activate GPR126 and trigger its downstream Gi signaling by binding to the ligand pocket in the seven-transmembrane domain of the C-terminal fragment of GPR126. A detailed mutagenesis screening according to a computational simulated structure model indicated that K1001ECL2 and F1012ECL2 are key residues that specifically recognize 17OHP but not progesterone. Finally, functional analysis revealed that progesterone-triggered GPR126 activation promoted cell growth in vitro and tumorigenesis in vivo, which involved Gi-SRC pathways in a triple-negative breast cancer model. Collectively, our work identified a membrane receptor for progesterone/17OHP and delineated the mechanisms by which GPR126 participated in potential tumor progression in triple-negative breast cancer, which will enrich our understanding of the functions and working mechanisms of both the aGPCR member GPR126 and the steroid hormone progesterone.

Structural basis for the tethered peptide activation of adhesion GPCRs.

Adhesion G-protein-coupled receptors (aGPCRs) are important for organogenesis, neurodevelopment, reproduction and other processes1-6. Many aGPCRs are activated by a conserved internal (tethered) agonist sequence known as the Stachel sequence7-12. Here, we report the cryogenic electron microscopy (cryo-EM) structures of two aGPCRs in complex with Gs: GPR133 and GPR114. The structures indicate that the Stachel sequences of both receptors assume an α-helical-bulge-ß-sheet structure and insert into a binding site formed by the transmembrane domain (TMD). A hydrophobic interaction motif (HIM) within the Stachel sequence mediates most of the intramolecular interactions with the TMD. Combined with the cryo-EM structures, biochemical characterization of the HIM motif provides insight into the cross-reactivity and selectivity of the Stachel sequences. Two interconnected mechanisms, the sensing of Stachel sequences by the conserved 'toggle switch' W6.53 and the constitution of a hydrogen-bond network formed by Q7.49/Y7.49 and the P6.47/V6.47φφG6.50 motif (φ indicates a hydrophobic residue), are important in Stachel sequence-mediated receptor activation and Gs coupling. Notably, this network stabilizes kink formation in TM helices 6 and 7 (TM6 and TM7, respectively). A common Gs-binding interface is observed between the two aGPCRs, and GPR114 has an extended TM7 that forms unique interactions with Gs. Our structures reveal the detailed mechanisms of aGPCR activation by Stachel sequences and their Gs coupling.

Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4.

Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and ß subunits1-3. A tethered agonism mediated by the 'Stachel sequence' of the ß subunit has been proposed to have central roles in aGPCR activation4-6. Here we present three cryo-electron microscopy structures of aGPCRs coupled to the Gs heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences-the ADGRG2-ß-Gs complex and the ADGRG4-ß-Gs complex (in which ß indicates the ß subunit of the aGPCR)-and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)-IP15-Gs). The Stachel sequences of both ADGRG2-ß and ADGRG4-ß assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-ß or ADGRG4-ß extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)-IP15-Gs complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM-p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-ß structures. By converting the 'finger residues' to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs.

Autonomous sensing of the insulin peptide by an olfactory G protein-coupled receptor modulates glucose metabolism.

Along with functionally intact insulin, diabetes-associated insulin peptides are secreted by ß cells. By screening the expression and functional characterization of olfactory receptors (ORs) in pancreatic islets, we identified Olfr109 as the receptor that detects insulin peptides. The engagement of one insulin peptide, insB:9-23, with Olfr109 diminished insulin secretion through Gi-cAMP signaling and promoted islet-resident macrophage proliferation through a ß cell-macrophage circuit and a ß-arrestin-1-mediated CCL2 pathway, as evidenced by ß-arrestin-1-/- mouse models. Systemic Olfr109 deficiency or deficiency induced by Pdx1-Cre+/-Olfr109fl/fl specifically alleviated intra-islet inflammatory responses and improved glucose homeostasis in Akita- and high-fat diet (HFD)-fed mice. We further determined the binding mode between insB:9-23 and Olfr109. A pepducin-based Olfr109 antagonist improved glucose homeostasis in diabetic and obese mouse models. Collectively, we found that pancreatic ß cells use Olfr109 to autonomously detect self-secreted insulin peptides, and this detection arrests insulin secretion and crosstalks with macrophages to increase intra-islet inflammation.

S-nitrosylation-mediated coupling of G-protein alpha-2 with CXCR5 induces Hippo/YAP-dependent diabetes-accelerated atherosclerosis.

Atherosclerosis-associated cardiovascular disease is one of the main causes of death and disability among patients with diabetes mellitus. However, little is known about the impact of S-nitrosylation in diabetes-accelerated atherosclerosis. Here, we show increased levels of S-nitrosylation of guanine nucleotide-binding protein G(i) subunit alpha-2 (SNO-GNAI2) at Cysteine 66 in coronary artery samples from diabetic patients with atherosclerosis, consistently with results from mice. Mechanistically, SNO-GNAI2 acted by coupling with CXCR5 to dephosphorylate the Hippo pathway kinase LATS1, thereby leading to nuclear translocation of YAP and promoting an inflammatory response in endothelial cells. Furthermore, Cys-mutant GNAI2 refractory to S-nitrosylation abrogated GNAI2-CXCR5 coupling, alleviated atherosclerosis in diabetic mice, restored Hippo activity, and reduced endothelial inflammation. In addition, we showed that melatonin treatment restored endothelial function and protected against diabetes-accelerated atherosclerosis by preventing GNAI2 S-nitrosylation. In conclusion, SNO-GNAI2 drives diabetes-accelerated atherosclerosis by coupling with CXCR5 and activating YAP-dependent endothelial inflammation, and reducing SNO-GNAI2 is an efficient strategy for alleviating diabetes-accelerated atherosclerosis.

PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates.

Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrate that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses reveal key residues that govern the interaction between PTP-MEG2 and its substrate, a peptide containing the phosphorylated NSF-pY83 site, specify PTP-MEG2 substrate selectivity, and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface reveals that PTP-MEG2 controls the fusion pore opening through NSF independent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we uncover that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 sites. Further structural and biochemical analyses confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies thus provide mechanistic insights in complex exocytosis processes.

Optimization of a peptide ligand for the adhesion GPCR ADGRG2 provides a potent tool to explore receptor biology.

The adhesion GPCR ADGRG2, also known as GPR64, is a critical regulator of male fertility that maintains ion/pH homeostasis and CFTR coupling. The molecular basis of ADGRG2 function is poorly understood, in part because no endogenous ligands for ADGRG2 have been reported, thus limiting the tools available to interrogate ADGRG2 activity. It has been shown that ADGRG2 can be activated by a peptide, termed p15, derived from its own N-terminal region known as the Stachel sequence. However, the low affinity of p15 limits its utility for ADGRG2 characterization. In the current study, we used alanine scanning mutagenesis to examine the critical residues responsible for p15-induced ADGRG2 activity. We next designed systematic strategies to optimize the peptide agonist of ADGRG2, using natural and unnatural amino acid substitutions. We obtained an optimized ADGRG2 Stachel peptide T1V/F3Phe(4-Me) (VPM-p15) that activated ADGRG2 with significantly improved (>2 orders of magnitude) affinity. We then characterized the residues in ADGRG2 that were important for ADGRG2 activation in response to VPM-p15 engagement, finding that the toggle switch W6.53 and residues of the ECL2 region of ADGRG2 are key determinants for VPM-p15 interactions and VPM-p15-induced Gs or arrestin signaling. Our study not only provides a useful tool to investigate the function of ADGRG2 but also offers new insights to guide further optimization of Stachel peptides to activate adhesion GPCR members.

Inhibition of striatal-enriched protein tyrosine phosphatase by targeting computationally revealed cryptic pockets.

Cryptic pockets, which are not apparent in crystallographic structures, provide promising alternatives to traditional binding sites for drug development. However, identifying cryptic pockets is extremely challenging and the therapeutic potential of cryptic pockets remains unclear. Here, we reported the discovery of novel inhibitors for striatal-enriched protein tyrosine phosphatase (STEP), a potential drug target for multiple neuropsychiatric disorders, based on cryptic pocket detection. By combining the use of molecular dynamics simulations and fragment-centric topographical mapping, we identified transiently open cryptic pockets and identified 12 new STEP inhibition scaffolds through structure-based virtual screening. Site-directed mutagenesis verified the binding of ST3 with the predicted cryptic pockets. Moreover, the most potent and selective inhibitors could modulate the phosphorylation of both ERK1/2 and Pyk2 in PC12 cells.

Deletion of pancreatic ß-cell adenosine kinase improves glucose homeostasis in young mice and ameliorates streptozotocin-induced hyperglycaemia.

Severe reduction in the ß-cell number (collectively known as the ß-cell mass) contributes to the development of both type 1 and type 2 diabetes. Recent pharmacological studies have suggested that increased pancreatic ß-cell proliferation could be due to specific inhibition of adenosine kinase (ADK). However, genetic evidence for the function of pancreatic ß-cell ADK under physiological conditions or in a pathological context is still lacking. In this study, we crossed mice carrying LoxP-flanked Adk gene with Ins2-Cre mice to acquire pancreatic ß -cell ADK deficiency (Ins2-Cre± Adkfl/fl ) mice. Our results revealed that Ins2-Cre+/- Adkfl/fl mice showed improved glucose metabolism and ß-cell mass in younger mice, but showed normal activity in adult mice. Moreover, Ins2-Cre± Adkfl/fl mice were more resistant to streptozotocin (STZ) induced hyperglycaemia and pancreatic ß-cell damage in adult mice. In conclusion, we found that ADK negatively regulates ß-cell replication in young mice as well as under pathological conditions, such as STZ induced pancreatic ß-cell damage. Our study provided genetic evidence that specific inhibition of pancreatic ß-cell ADK has potential for anti-diabetic therapy.

Structural Mechanism of the Arrestin-3/JNK3 Interaction.

Arrestins, in addition to desensitizing GPCR-induced G protein activation, also mediate G protein-independent signaling by interacting with various signaling proteins. Among these, arrestins regulate MAPK signal transduction by scaffolding mitogen-activated protein kinase (MAPK) signaling components such as MAPKKK, MAPKK, and MAPK. In this study, we investigated the binding mode and interfaces between arrestin-3 and JNK3 using hydrogen/deuterium exchange mass spectrometry, 19F-NMR, and tryptophan-induced Atto 655 fluorescence-quenching techniques. Results suggested that the ß1 strand of arrestin-3 is the major and potentially only interaction site with JNK3. The results also suggested that C-lobe regions near the activation loop of JNK3 form the potential binding interface, which is variable depending on the ATP binding status. Because the ß1 strand of arrestin-3 is buried by the C-terminal strand in its basal state, C-terminal truncation (i.e., pre-activation) of arrestin-3 facilitates the arrestin-3/JNK3 interaction.

Computational Strategy for Bound State Structure Prediction in Structure-Based Virtual Screening: A Case Study of Protein Tyrosine Phosphatase Receptor Type O Inhibitors.

Accurate protein structure in the ligand-bound state is a prerequisite for successful structure-based virtual screening (SBVS). Therefore, applications of SBVS against targets for which only an apo structure is available may be severely limited. To address this constraint, we developed a computational strategy to explore the ligand-bound state of a target protein, by combined use of molecular dynamics simulation, MM/GBSA binding energy calculation, and fragment-centric topographical mapping. Our computational strategy is validated against low-molecular weight protein tyrosine phosphatase (LMW-PTP) and then successfully employed in the SBVS against protein tyrosine phosphatase receptor type O (PTPRO), a potential therapeutic target for various diseases. The most potent hit compound GP03 showed an IC50 value of 2.89 µM for PTPRO and possessed a certain degree of selectivity toward other protein phosphatases. Importantly, we also found that neglecting the ligand energy penalty upon binding partially accounts for the false positive SBVS hits. The preliminary structure-activity relationships of GP03 analogs are also reported.

Arctigenin Attenuates Ischemia/Reperfusion Induced Ventricular Arrhythmias by Decreasing Oxidative Stress in Rats.

BACKGROUND/AIMS: Arctigenin (ATG) has been shown to possess anti-inflammatory, immunemodulatory, anti-viral, anti-microbial, anti-carcinogenic, vasodilatory and anti-platelet aggregation properties. However, the protective role of ATG in prevention of arrhythmias induced by myocardial ischemia/reperfusion is unknown. The aim of this study was to investigate the anti-arrhythmia effect of ATG in an ischemia/reperfusion injured rat heart model and explore the related mechanisms. METHODS: Rats were randomly exposed to sham operation, myocardial ischemia/ reperfusion (MI/R) alone, ATG+ MI/R, pretreated with ATG in low (12.5 mg/kg/day), medium (50 mg/kg/day) and high dose (200 mg/kg/day), respectively. Ventricular arrhythmias were assessed. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the level of malondialdehyde (MDA) in myocardial tissue were determined by chemical analysis. RESULTS: Compared to MI/R, rats pretreated with ATG in doses of 50 mg/kg/day and 200 mg/kg/day showed significantly reduced incidence and duration of ventricular fibrillation, ventricular tachycardia and ventricular ectopic beat (VEB), and decreased the arrhythmia score during the 30-min ischemia. Incidence and duration of ventricular tachycardia, infarction size and arrhythmia scores in these groups were significantly decreased during the 120-min reperfusion. No ventricular fibrillation occurred during the period of reperfusion. Rats pretreated with ATG in doses of 50 mg/kg/day and 200 mg/kg/ day markedly enhanced the activities of antioxidant enzymes SOD and GSH-Px, reduced the level of MDA. No differences were observed between the group pretreated with a low dose of ATG and the sham group. Administration of ATG significantly increased the expression of antioxidant stress protein Nrf2, Trx1 and Nox1. CONCLUSION: Our data suggested that ATG plays anti-arrhythmia role in ischemia/reperfusion injury, which is probably associated with attenuating oxidative stress by Nrf2 signaling pathway.

Gq activity- and ß-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility.

Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and ß-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or ß-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that ß-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/ß-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility.

Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

The protein tyrosine phosphatase nonreceptor type 12 (PTPN12) is a multifunctional protein and has elicited much research attention because its decreased protein level has been associated with poor prognosis of several types of cancers. Recently, we have solved the crystal structure of the phosphatase domain of PTPN12, which disclosed a specific PTPN12-insert-loop harboring a cyclin-dependent kinase 2 (CDK2) phosphorylation site. However, the functional significance of this phosphorylation is undefined. In the present study, we found that S19 site phosphorylation of PTPN12 by CDK2 discharged its antitumor activity by down-regulation of its inhibitory role in cell migration, but not affecting its other regulatory functions. Phosphorylation of PTPN12 at the S19 site changed its substrate interface, and by doing so, selectively decreased its activity toward the human epidermal growth factor receptor 2 (HER2)- pY1196 site, but not other HER2 phosphorylation sites or other known PTPN12 substrates. A further in-depth mechanism study revealed that the phosphorylation of PTPN12 by CDK2 impaired recruitment of the serine/threonine-protein kinase 1 (PAK1) to HER2, resulted in the blockade of the HER2-pY1196-PAK1-T423 signaling pathway, thus increased tumor cell motility. Taken together, our results identified a new phosphorylation-based substrate recognition mechanism of PTPN12 by CDK2, which orchestrated signaling crosstalk between the oncogenic CDK2 and HER2 pathways. The newly identified governing mechanism of the substrate selectivity of a particular phosphatase was previously unappreciated and exemplifies how a phospho-network is precisely controlled in different cellular contexts.-Li, H., Yang, D., Ning, S., Xu, Y., Yang, F., Yin, R., Feng, T., Han, S., Guo, L., Zhang, P., Qu, W., Guo, R., Song, C., Xiao, P., Zhou, C., Xu, Z., Sun, J.-P., Yu, X. Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

A cullin 4B-RING E3 ligase complex fine-tunes pancreatic δ cell paracrine interactions.

Somatostatin secreted by pancreatic δ cells mediates important paracrine interactions in Langerhans islets, including maintenance of glucose metabolism through the control of reciprocal insulin and glucagon secretion. Disruption of this circuit contributes to the development of diabetes. However, the precise mechanisms that control somatostatin secretion from islets remain elusive. Here, we found that a super-complex comprising the cullin 4B-RING E3 ligase (CRL4B) and polycomb repressive complex 2 (PRC2) epigenetically regulates somatostatin secretion in islets. Constitutive ablation of CUL4B, the core component of the CRL4B-PRC2 complex, in δ cells impaired glucose tolerance and decreased insulin secretion through enhanced somatostatin release. Moreover, mechanistic studies showed that the CRL4B-PRC2 complex, under the control of the δ cell-specific transcription factor hematopoietically expressed homeobox (HHEX), determines the levels of intracellular calcium and cAMP through histone posttranslational modifications, thereby altering expression of the Cav1.2 calcium channel and adenylyl cyclase 6 (AC6) and modulating somatostatin secretion. In response to high glucose levels or urocortin 3 (UCN3) stimulation, increased expression of cullin 4B (CUL4B) and the PRC2 subunit histone-lysine N-methyltransferase EZH2 and reciprocal decreases in Cav1.2 and AC6 expression were found to regulate somatostatin secretion. Our results reveal an epigenetic regulatory mechanism of δ cell paracrine interactions in which CRL4B-PRC2 complexes, Cav1.2, and AC6 expression fine-tune somatostatin secretion and facilitate glucose homeostasis in pancreatic islets.

A prediction model for lymph node metastases using pathologic features in patients intraoperatively diagnosed as stage I non-small cell lung cancer.

BACKGROUND: There is little information on which pattern should be chosen to perform lymph node dissection for stage I non-small-cell lung cancer. This study aimed to develop a model for predicting lymph node metastasis using pathologic features of patients intraoperatively diagnosed as stage I non-small-cell lung cancer. METHODS: We collected pathology data from 284 patients intraoperatively diagnosed as stage I non-small-cell lung cancer who underwent lobectomy with complete lymph node dissection from 2013 through 2014, assessing various factors for an association with metastasis to lymph nodes (age, gender, pathology, tumour location, tumour differentiation, tumour size, pleural invasion, bronchus invasion, multicentric invasion and angiolymphatic invasion). After analysing these variables, we developed a multivariable logistic model to estimate risk of metastasis to lymph nodes. RESULTS: Univariate logistic regression identified tumour size >2.65 cm (p < 0.001), tumour differentiation (p < 0.001), pleural invasion (p = 0.034) and bronchus invasion (p < 0.001) to be risk factors significantly associated with the presence of metastatic lymph nodes. On multivariable analysis, only tumour size >2.65 cm (p < 0.001), tumour differentiation (p = 0.006) and bronchus invasion (p = 0.017) were independent predictors for lymph node metastasis. We developed a model based on these three pathologic factors that determined that the risk of metastasis ranged from 3% to 44% for patients intraoperatively diagnosed as stage I non-small-cell lung cancer. By applying the model, we found that the values y > 0.80, 0.43 < y ≤ 0.80, y ≤ 0.43 plus tumour size >2 cm and y ≤0.43 plus tumour size ≤2 cm yielded positive lymph node metastasis predictive values of 44%, 18%, 14% and 0%, respectively. CONCLUSIONS: A non-invasive prediction model including tumour size, tumour differentiation and bronchus invasion may be useful to give thoracic surgeons recommendations on lymph node dissection for patients intraoperatively diagnosed as Stage I non-small cell lung cancer.