Signaling Profiles Research Articles

Human placental mesenchymal stromal cell therapy restores the cytokine efflux and insulin signaling in the skeletal muscle of obesity-induced type 2 diabetes rat model.

Obesity poses a significant risk factor for the onset of metabolic syndrome with allied complications, wherein mesenchymal stem cell therapy is seen as a promising treatment for obesity-induced metabolic syndrome. In the present study, we aim to explore the beneficial effects of the human placental mesenchymal stromal cells (P-MSCs) on obesity-associated insulin resistance (IR) including inflammation. To understand this, we have analyzed the peripheral blood glucose, serum insulin levels by ELISA, and the glucose uptake capacity of skeletal muscle by a 2-NBDG assay using flow cytometry in WNIN/GR-Ob rats treated with and without P-MSCs. Also, we have studied insulin signaling and cytokine profile in the skeletal muscle by western blotting, dot blotting, and Multiplex-ELISA techniques. The skeletal muscle of WNIN/GR-Ob rats demonstrates dysregulation of cytokines, altered glucose uptake vis-a-vis insulin signaling. However, P-MSCs' treatment was effective in WNIN/GR-Ob rats as compared to its control, to restore HOMA-IR, re-establishes dysregulated cytokines and PI3K-Akt pathway in addition to enhanced Glut4 expression and glucose uptake studied in skeletal muscle. Overall, our data advocate the beneficial effects of P-MSCs to ameliorate inflammatory milieu, improve insulin sensitivity, and normalize glucose homeostasis underlining the Ob-T2D conditions, and we attribute for immunomodulatory, paracrine, autocrine, and multipotent functions of P-MSCs.

Regulation of microRNAs in Alzheimer´s disease, type 2 diabetes, and aerobic exercise training.

Alzheimer's disease (AD) is the most common type of dementia. The evolution and aggregation of amyloid beta (β) oligomers is linked to insulin resistance in AD, which is also the major characteristic of type 2 diabetes (T2D). Being physically inactive can contribute to the development of AD and/or T2D. Aerobic exercise training (AET), a type of physical exercise, can be useful in preventing or treating the negative outcomes of AD and T2D. AD, T2D and AET can regulate the expression of microRNAs (miRNAs). Here, we review some of the changes in miRNAs expression regulated by AET, AD and T2D. MiRNAs play an important role in the gene regulation of key signaling pathways in both pathologies, AD and T2D. MiRNA dysregulation is evident in AD and has been associated with several neuropathological alterations, such as the development of a reactive gliosis. Expression of miRNAs are associated with many pathophysiological mechanisms involved in T2D like insulin synthesis, insulin resistance, glucose intolerance, hyperglycemia, intracellular signaling, and lipid profile. AET regulates miRNAs levels. We identified 5 miRNAs (miR-21, miR-29a/b, miR-103, miR-107, and miR-195) that regulate gene expression and are modulated by AET on AD and T2D. The identified miRNAs are potential targets to treat the symptoms of AD and T2D. Thus, AET is a non-pharmacological tool that can be used to prevent and fight the negative outcomes in AD and T2D.

Dopamine D2L receptor density influences the recruitment of β-arrestin2 and Gi1 induced by antiparkinsonian drugs

IntroductionBrain imaging studies have highlighted that the density of dopamine D2 receptors markedly fluctuates across the stages of Parkinson's disease and in response to pharmacological treatment. Moreover, receptor density constitutes a molecular determinant for the signaling profile of D2 receptor ligands. We therefore hypothesized that variations in receptor expression could influence D2 receptor response to antiparkinsonian drugs, most notably with respect to the recruitment bias between Gi1 and β-arrestin2. MethodsThe recruitment bias of dopamine, pramipexole, ropinirole, and rotigotine was examined using a nanoluciferase-based biosensor for probing the interactions of the D2L receptor with either Gi1 or β-arrestin2. The characterization of the functional selectivity of these D2 receptor agonists was performed at two distinct D2L receptor densities by taking advantage of a cell model carrying an inducible system that enables the overexpression of the D2L receptor when exposed to doxycycline. ResultsA high receptor density oriented the balanced signaling profile of dopamine towards a preferential recruitment of Gi1. It also moderated the marked Gi1 and β-arrestin2 biases of pramipexole and rotigotine, respectively. At variance, the Gi1 bias of ropinirole appeared as not being influenced by D2L receptor density. ConclusionsTaken together, these observations highlight receptor density as a key driver of the signaling transducer recruitment triggered by antiparkinsonian agents. Moreover, given the putative beneficial properties of β-arrestin2 in promoting locomotion, this study provides molecular insights that position the arrestin-biased ligand rotigotine as a putatively more beneficial D2 receptor agonist for the treatment of early and late Parkinson's disease.

Galanin enhanced insulin-mediated intracellular signaling by regulating the stability of membrane-localized insulin/IR.

Previous studies have shown that insulin has the important regulatory effect on the intestinal tract. However, until now, the biological properties of insulin on intestinal cell has not been revealed. Therefore, in the current research, we first studied the cell characteristics and signaling profiles of insulin in the intestinal cell model, and found that insulin can be internalized into the cytoplasm in a time-dependent manner. After internalization, insulin transported into different type of endosomes. More importantly, we explored the effect of galanin on insulin-mediated signaling pathways (galanin is a polypeptide composed of 29 amino acid residues, galanin is widely distributed in the central and peripheral nervous system and has a variety of biological activities), and found that galanin can increase insulin sensitivity by regulating insulin receptor (IR)-mediated signal transduction pathways. We further study the potential molecular mechanism by which galanin enhances insulin sensitivity, and found that galanin could increase the time of insulin acting on the cell membrane. Further experiments showed that galanin could stabilize the membrane-localized insulin/IR, which may be an important new potential mechanism by which galanin improves the biological activity of insulin. This study laid the foundation for exploring the relationship between galanin and insulin sensitivity.

One amino acid mutation of prostanoid EP4 receptor altered its signaling profiles.

One amino acid mutation of prostanoid EP4 receptor altered its signaling profiles.

Proteogenomic characterization identifies clinically relevant subgroups of intrahepatic cholangiocarcinoma.

We performed proteogenomic characterization of intrahepatic cholangiocarcinoma (iCCA) using paired tumor and adjacent liver tissues from 262 patients. Integrated proteogenomic analyses prioritized genetic aberrations and revealed hallmarks of iCCA pathogenesis. Aflatoxin signature was associated with tumor initiation, proliferation, and immune suppression. Mutation-associated signaling profiles revealed that TP53 and KRAS co-mutations may contribute to iCCA metastasis via the integrin-FAK-SRC pathway. FGFR2 fusions activated the Rho GTPase pathway and could be a potential source of neoantigens. Proteomic profiling identified four patient subgroups (S1-S4) with subgroup-specific biomarkers. These proteomic subgroups had distinct features in prognosis, genetic alterations, microenvironment dysregulation, tumor microbiota composition, and potential therapeutics. SLC16A3 and HKDC1 were further identified as potential prognostic biomarkers associated with metabolic reprogramming of iCCA cells. This study provides a valuable resource for researchers and clinicians to further identify molecular pathogenesis and therapeutic opportunities in iCCA.

Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

Frizzleds (FZD1–10) are G protein–coupled receptors containing an extracellular cysteine-rich domain (CRD) binding Wingless/Int-1 lipoglycoproteins (WNTs). Despite the role of WNT/FZD signaling in health and disease, our understanding of how WNT binding is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics. To clarify how WNT binding to FZD translates into receptor dynamics, we devised conformational FZD-CRD biosensors based on bioluminescence resonance energy transfer (BRET). Using FZD with N-terminal nanoluciferase (Nluc) and fluorescently labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct signaling pathways and that CRD dynamics are not required for WNT/β-catenin signaling. Thus, these FZD-CRD biosensors provide insights into binding, activation, and signaling processes in FZDs. The sensor design is broadly applicable to explore ligand-induced dynamics also in other membrane receptors.

Glucocorticoid-Resistant B-Cell Acute Lymphoblastic Leukemic Cells Can be Targeted By BCR-Signaling Inhibition

Glucocorticoids (GCs) remain a backbone component of therapeutic regimens for childhood B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is a strong prognostic marker of relapse making its understanding an important challenge to be addressed to improve overall patients outcome.Healthy B-cell development is characterized by checkpoints where critical regulatory signaling influences the fate of developing B-cells. These stages are vulnerable for leukemic transformation and as we previously demonstrated, the developmental and functional state of B-ALL cells are of critical importance in treatment failure. Using this developmental framework, we examined the effect of GCs on healthy and malignant B-cells to better understand mediators of GC resistance and ways to overcome it.To define the dynamics of the transcriptional networks surrounding B-cell developmental checkpoints, we performed transcriptomic analysis of sorted pre-proB, pro-B and pre-B cells from 3 healthy donors. We found a coordinated upregulation of B cell receptor (BCR) and Glucocorticoid Receptor (GCR) pathways in healthy B cells during their development. Single cell proteomic analysis of these healthy populations confirmed the coordinated expression of GCR with early B-cell phenotypic markers. Furthermore, in vitro treatment of healthy B-cells with the GC dexamethasone (dex) demonstrated cycling pro-B/pre-B cells to be the most sensitive to GC-induced cell death.Given the importance of GCs in B-ALL treatment, we investigated GCs effects and resistance in NALM6 and REH cell lines. NALM6, expressing high levels of GCR, were significantly sensitive to dex treatment in terms of cell cycle arrest and cell death. By contrast, REH cells were resistant to dex treatment as they lack the GCR. Retroviral transfection of NR3C1 (GCR gene) in REH cells (REH GCR) resulted in acquired sensitivity to dex. To explore the potential crosstalk between GCR and BCR pathways, we also treated cells with BCR signaling inhibitor, dasatinib (das), alone or in combination with dex. While both cell lines survived to treatment with das, the combined treatment increased apoptosis compared to dex alone in NALM6 cells (p=0.0179) and REH GCR cells (p=ns). Whole transcriptome sequencing of dex-resistant cells revealed upregulation of BCR downstream signaling as one of the main pathways associated with resistance.The cell line data implicated active BCR signaling as a path to GCs resistance, so we next analyzed 19 B-ALL primary samples by mass cytometry, after in vitro exposure for 48 hrs to same treatments. Across the entire cohort, dex induced a significant reduction in viability (p=0.0007), compared to vehicle. Treatment with das and dex+das also decreased cell viability compared to vehicle (p=0.0038 and p=0.0002) although was not significant to dex alone. Interestingly dex-resistant cells showed a phenotypic modulation compatible with a late pre-B phenotype with increased CD45 and CD20 expression. In addition, surviving cells showed an activation of downstream targets of BCR signaling such as pSYK, pRPS6 and pCREB that was partially blunted by dasatinib treatment.To understand whether the phenotype and signaling modulation induced by dexamethasone also occurs in patients after treatment with GCs, we analyzed minimal residual disease (MRD) cells following 8 days of treatment with GCs from 9 B-ALL patients. This analysis confirmed our previous findings with MRD cells having same late pre-B cells phenotype and signaling profile as in vitro treated cells.Finally, we tested whether targeting of pre-BCR signaling via dasatinib, could overcome resistance in vivo. We evaluated engraftment of luciferase-expressing NALM6 cells at different timepoints after tail vein injection in mice treated with vehicle, dex, das or dex + das. Bioluminescence analysis revealed a significant reduction of early and late engraftment in the dex + das group compared to vehicle-treated mice. Furthermore, mice receiving the combined treatment also experienced a significant survival advantage as assessed by log rank test (p=0.0002).Taken together these data suggest a coordinated interplay between BCR and GCR pathways in healthy and leukemic B cells. GCs-resistant leukemic cells showed a mature pre-B phenotype that is vulnerable to BCR signaling inhibition in vitro and in vivo suggesting new therapeutic options to overcome GC resistance in childhood B-ALL. DisclosuresBiondi: Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria. Bava: 10x Genomics: Current Employment. Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

Distinctive Signaling Profiles With Distinct Biological and Clinical Implications in Aggressive CLL Subsets With Stereotyped B-Cell Receptor Immunoglobulin.

The ontogeny and evolution of chronic lymphocytic leukemia (CLL) are critically dependent on interactions between leukemic cells and their microenvironment, including antigens, the latter recognized through the clonotypic B-cell receptor immunoglobulin (BcR IG). Antigen selection is key to the pathogenesis of CLL, as evidenced by the remarkable skewing of the BcR IG gene repertoire, culminating in BcR IG stereotypy, referring to the existence of subsets of patients with (quasi)identical BcR IG. Notably, certain of these subsets have been found to display distinct, subset-biased biological background, clinical presentation, and outcome, including the response to treatment. This points to BcR IG centrality while also emphasizing the need to dissect the signaling pathways triggered by the distinctive BcR IG expressed by different subsets, particularly those with aggressive clinical behavior. In this mini-review, we discuss the current knowledge on the implicated signaling pathways as well as the recurrent gene mutations in these pathways that characterize major aggressive stereotyped subsets. Special emphasis is given on the intertwining of BcR IG and Toll-like receptor (TLR) signaling and the molecular characterization of signaling activation, which has revealed novel players implicated in shaping clinical aggressiveness in CLL, e.g., the histone methyltransferase EZH2 and the transcription factor p63.

Characterisation of agonist signalling profiles and agonist-dependent antagonism at PACAP-responsive receptors: Implications for drug discovery.

The pituitary adenylate cyclase-activating peptide (PACAP) family is of clinical interest for the treatment of migraine. These peptides activate three different PACAP-responsive class B G protein-coupled receptors: the PAC1 , VPAC1 and VPAC2 receptors. The PAC1 receptor may be alternatively spliced, generating variants that can differ in their pharmacological or signalling profiles. To inform drug discovery efforts targeting migraine, we need to better understand how the different PACAP-responsive receptors signal and how effectively these responses can be blocked by antagonists. The signalling profiles of the human PAC1n , PAC1s , VPAC1 and VPAC2 receptors were examined in transfected Cos7 cells for cAMP, IP1 , pAkt, pERK and pCREB. Biased signalling was then quantified. The ability of antagonists to block PACAP-38, PACAP-27 or VIP stimulated cAMP accumulation at PACAP-responsive receptors was also determined. PACAP-responsive receptors exhibited varied pharmacological profiles but activated signalling in a similar manner. The PAC1n and PAC1s receptors displayed distinct pharmacology. At the PAC1s receptor, VIP and PHM were more potent than at the PAC1n receptor. PACAP-responsive receptors displayed agonist-dependent antagonism where PACAP-38 was less effectively antagonised compared to PACAP-27 and VIP. The distinct pharmacological profile displayed by the PAC1s receptor suggests that it can act as a dual receptor for VIP and PACAP. Furthermore, the effectiveness of blocking a signalling pathway can be influenced by which endogenous PACAP family agonist is present. These effects have potential implications for the development and effectiveness of drugs targeting the PACAP system. This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc.

Mutated JAK2 signal transduction in human induced pluripotent stem cell (iPSC)-derived megakaryocytes

Janus kinase 2 (JAK2) gene mutations are the main drivers for polycythemia vera (PV) and essential thrombocythemia (ET). The mechanisms of single altered gene causing two different diseases are unclear. Additionally, novel treatments specifically targeting mutated JAK2 proteins are needed. In this study, the induced pluripotent stem cells (iPSCs) were virally transduced to express wild-type JAK2 (JAK2WT), JAK2p.V617F (JAK2V617F) or JAK2p.N542_E543del (JAK2exon12) under a doxycycline-inducible system. The modified iPSCs which were differentiated into megakaryocytes in the presence vs. absence of doxycycline were compared to ensure that the differences were solely from mutated JAK2 expressions. The JAK2V617-expressing iPSCs yielded significantly higher numbers of megakaryocytes consistent with the ET phenotype, while there was no enhancement by JAK2exon12 expression compatible with the pure erythrocytosis in humans. Capillary Western analyses revealed significantly greater JAK2 phosphorylation in iPSCs carrying JAK2V617F but not in JAK2WT and JAK2exon12 iPSCs. Activation of STAT3, STAT5 and AKT was increased by JAK2V617F, while they were decreased in JAK2exon12 iPSCs. Notably, interferon alpha and/or arsenic trioxide inhibited megakaryocytes proliferation and reduced JAK2, STAT3, STAT5 and AKT phosphorylation in mutant JAK2-expressing iPSCs compared with those without induction. In conclusion, JAK2V617F expression in iPSCs preferentially promoted megakaryocytes with a signaling profile distinctive from JAK2exon12 expression. Treatments with interferon alpha or arsenic trioxide preferentially suppressed the mutated over wild-type JAK2 signaling. This iPSC model is helpful in mechanistic studies and novel therapy screen for myeloproliferative neoplasm.

Orthogonal Peptide-Templated Labeling Elucidates Lateral ETA R/ETB R Proximity and Reveals Altered Downstream Signaling.

Fine‐tuning of G protein‐coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one‐step labeling method of multiple membrane‐embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ETAR and ETBR), angiotensin II receptor type 1, and apelin. Wild type‐like G protein activation after N‐terminal modification was demonstrated for all receptor species. Using FRET‐competent dyes, a constitutive proximity between hetero‐receptors was limited to ETAR/ETBR. Further, we demonstrate, that ETAR expression regulates the signaling of co‐expressed ETBR. Our orthogonal peptide‐templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.

Primary vulvar squamous cell carcinomas with high T cell infiltration and active immune signaling are potential candidates for neoadjuvant PD-1/PD-L1 immunotherapy

BackgroundA profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture, signaling pathways and checkpoint...

64P Tyrosine kinase signaling profile in osteosarcomas: A potential therapeutic target for sensitization to doxorubicin

Metastatic and recurrent forms of osteosarcoma (OS) are characterized by aggressive behaviour, resistance to the current chemotherapeutic regimens, and an extremely poor prognosis. Therefore, the identification of specific markers of disease progression and sensitivity to the chemotherapeutic agents remains a prior goal for molecular and genetic research in precision oncology.

Ackr3-Venus knock-in mouse lights up brain vasculature

The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits βarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.

Allosteric Modulators Enhancing GLP-1 Binding to GLP-1R via a Transmembrane Site.

The glucagon-like peptide-1 receptor (GLP-1R) is a well-established drug target for the treatment of type II diabetes. The development of small-molecule positive allosteric modulators (PAMs) of GLP-1R is a promising therapeutic strategy. Here, we report the discovery and characterization of PAMs with distinct chemotypes, binding to a cryptic pocket formed by the cytoplasmic half of TM3, TM5, and TM6. Molecular dynamic simulations and mutagenesis studies indicate that the PAM enlarges the orthosteric pocket to facilitate GLP-1 binding. Further signaling assays characterized their probe-dependent signaling profiles. Our findings provide mechanistic insights into fine-tuning GLP-1R via this allosteric pocket and open up new avenues to design small-molecule drugs for class B G-protein-coupled receptors.

Tofacitinib Suppresses Several JAK-STAT Pathways in Rheumatoid Arthritis In Vivo and Baseline Signaling Profile Associates With Treatment Response.

ObjectiveCurrent knowledge on the actions of tofacitinib on cytokine signaling pathways in rheumatoid arthritis (RA) is based on in vitro studies. Our study is the first to examine the effects of tofacitinib treatment on Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathways in vivo in patients with RA.MethodsSixteen patients with active RA, despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), received tofacitinib 5 mg twice daily for three months. Levels of constitutive and cytokine-induced phosphorylated STATs in peripheral blood monocytes, T cells and B cells were measured by flow cytometry at baseline and three-month visits. mRNA expression of JAKs, STATs and suppressors of cytokine signaling (SOCS) were measured from peripheral blood mononuclear cells (PBMCs) by quantitative PCR. Association of baseline signaling profile with treatment response was also investigated.ResultsTofacitinib, in csDMARDs background, decreased median disease activity score (DAS28) from 4.4 to 2.6 (p < 0.001). Tofacitinib treatment significantly decreased cytokine-induced phosphorylation of all JAK-STAT pathways studied. However, the magnitude of the inhibitory effect depended on the cytokine and cell type studied, varying from 10% to 73% inhibition following 3-month treatment with tofacitinib. In general, strongest inhibition by tofacitinib was observed with STAT phosphorylations induced by cytokines signaling through the common-γ-chain cytokine receptor in T cells, while lowest inhibition was demonstrated for IL-10 -induced STAT3 phosphorylation in monocytes. Constitutive STAT1, STAT3, STAT4 and STAT5 phosphorylation in monocytes and/or T cells was also downregulated by tofacitinib. Tofacitinib treatment downregulated the expression of several JAK-STAT pathway components in PBMCs, SOCSs showing the strongest downregulation. Baseline STAT phosphorylation levels in T cells and monocytes and SOCS3 expression in PBMCs correlated with treatment response.ConclusionsTofacitinib suppresses multiple JAK-STAT pathways in cytokine and cell population specific manner in RA patients in vivo. Besides directly inhibiting JAK activation, tofacitinib downregulates the expression of JAK-STAT pathway components. This may modulate the effects of tofacitinib on JAK-STAT pathway activation in vivo and explain some of the differential findings between the current study and previous in vitro studies. Finally, baseline immunological markers associate with the treatment response to tofacitinib.

Structures of active melanocortin-4 receptor\u2013Gs-protein complexes with NDP-\u03b1-MSH and setmelanotide

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R–Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor–Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.

MiR-146a suppresses the expression of CXCR4 and alters survival, proliferation and migration rate in colorectal cancer cells

CXCR4 plays an important role in colorectal cancer (CRC) development and metastasis. Some previous studies have indicated CXCR4 as a therapeutic target in cancer. CXCR4 is known as a direct target of miR-146a. The present study aimed to investigate how exogenous induction of miR-146a affects CXCR4 gene and protein expression and also proliferation, apoptosis and migration of CRC cells. Transfection of Caco-2 and SW480 cells by a synthetic miR-146a mimic led to downregulation of CXCR4 expression at both gene and protein levels. It also downregulated expression of several miR-146a targets, including GSK3B, IRAK1, TRAF6, AKT2, SMAD4, EGFR and NFKB1, mostly in SW480 cells. Overexpression of miR-146a resulted in a partial cell cycle arrest in the both cell lines, while the apoptotic rate was also decreased. In regards to epithelial-mesenchymal transition factors, VIM was downregulated in the both cell lines, but SNAI1 was upregulated in Caco-2 cells. The wound closure assay showed a reduction in cell migration in SW480 cells, but an opposite effect was detected in Caco-2 cells following transfection with miR-146a mimic. Therefore, our results are indicating that overexpression of miR-146a, despite downregulation of oncogenic CXCR4, may not lead to a universal tumor suppressive effect in all CRC cells, and this is possibly due to differences in miR-146a effects on signaling pathways in each cell type. Selection of miR-146a for tumor suppression requires enough details regarding the signaling profile of cancer cells otherwise it may produce unexpected outcome.

Cryo-EM structure of the dual incretin receptor agonist, peptide-19, in complex with the glucagon-like peptide-1 receptor

Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.