Protein Activity In Response Research Articles

Abstract PO4-23-09: Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells

Abstract In HER2-enriched (HER2E) breast cancer (BC), the dual HER2/EGFR inhibitor Lapatinib is a standard of care for advanced staged disease. Anti-HER2 therapies disrupt tumor progression through inhibition of the PI3K/AKT pathway. Some treated tumors will override inhibition by shifting to MAPK/ERK signaling to fuel cell growth, thus fostering resistance to anti-HER2 therapy. The tyrosine receptor kinase FGFR4 is included in the profile for a HER2E subtype. FGFR4 is overexpressed in ~30% of BC and can signal through PI3K/AKT and MAPK/ERK. Inhibitors for FGFR4 such as BLU554 are being investigated in phase I/II clinical trials for hepatocellular carcinoma, showing success in tumors that are positive for FGFR4’s preferred ligand FGF19. According to cBioPortal patient data, FGF19 is amplified in ~20% of BC, and has been shown to trigger an autocrine loop in triple negative BC through FGFR4. We examined BLU554 as a dual therapeutic with Lapatinib and dissected intracellular signaling stemming from the combination in HER2E BC. MDA-MB-453 BC cells (HER2+FGFR4+) were used for a dual strategy with BLU554 and Lapatinib. Cell viability assays were performed at matched doses, then as a dose response matrix, ranging from 0.1-20 µM. Findings were used for synergy analysis using synergyfinder.fimm.fi. Western blots determined protein activity in response to treatments. Ridaforolimus (mTOR inhibitor) was used in combination with either BLU554 or Lapatinib to assess MAPK/ERK and PI3K/AKT pathway crosstalk in MB-453 cells. Additionally, FGF19 was added to cells after a 2-hour pre-treatment to challenge BLU554 + Lapatinib and compare changes in signaling with FGF19 present. MB-453 cells treated with BLU554 decreased cell viability similarly to Lapatinib at 10 µM (65% [SD ± 3.3] and 63% [SD ± 2.1], respectively). Dual treatment decreased viability compared to single agents at all concentrations but the greatest difference was observed at 20 µM (38.5% in dual compared to Lapatinib [p-value = 0.00006]; 45.1% in dual compared to BLU554 [p-value = 0.00016]). Dose-response matrix of dual treatment yielded a ZIP synergy score of 16.852 ± 2.97 (>10 is synergistic), with the greatest synergy and inhibition at 10 µM and 20 µM of both drugs. BLU554 caused a therapeutic potency shift of Lapatinib, such that lower doses of Lapatinib (0.1 µM, 1 µM) are effective at higher doses of BLU554 (10 µM, 20 µM). Westerns of MB-453 with one dose of BLU554 increased phospho-ERK1/2 (pERK1/2) at 16- or 48-hr time points versus control, but when BLU554 was re-dosed then pERK1/2 was successfully inhibited. Both pAKT and pS6RP were increased by re-dosing BLU554. Cells treated for 2 hrs with Lapatinib decreased pAKT and pS6RP and did not change pERK, whereas a 2-hr treatment of BLU554 increased pAKT and decreased pERK. Dual treatment exhibited inhibition of ERK, AKT, 4EBP1, and S6RP phosphorylation all to a greater extent than single agents in combination with Ridaforolimus. Challenge by ectopic FGF19 of BLU554 + Lapatinib was unable to restore downstream signaling of AKT and ERK. BLU554 synergistically interacts with Lapatinib to decrease cell viability while increasing its therapeutic potency. Lapatinib primarily inhibits AKT signaling, whereas BLU554 targets ERK. Dual treatment simultaneously blocks MAPK/ERK and PI3K/AKT signaling and yields decreases of mTOR downstream effectors pS6RP and p4EBP1. These effects are maintained even when challenged with FGF19, suggesting that dual inhibition of FGFR4 and HER2 could prevent pathway activation from external stimuli. Citation Format: Jordan Beardsley, Joseph Goode, Deborah Altomare. Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-09.

Modulation of phosphorylation status of MAP3 kinases under abiotic stress responses

AbstractPlants must face various environmental stresses that they cannot avoid, and they have developed numerous mechanisms to survive these challenges. Phosphate‐mediated signal transduction is a common method for regulating gene expression and protein activity in response to stresses. Phosphate groups can be transferred to other proteins by proteins called kinases. MAP3Ks are the largest kinase family in plants, and they comprise various group and individual pathways collectively known as MAPK cascades. When plants encounter environmental stresses, several MAP3Ks act as positive or negative regulators by phosphorylating downstream proteins within the MAPK cascade, as well as transcription factors, cytoskeletal proteins, enzymes, and kinases, through their kinase activity. In addition, other upstream kinases and phosphatases regulate MAP3Ks by adding or removing phosphate groups. Various MAP3Ks have been isolated in many plant species, and evidence suggests that they function as post‐translational modulators under various abiotic stresses, including ABA responses as well as salinity, osmotic, drought, oxidative, cold, and heat stress. This review focuses on how plant MAP3Ks affect various abiotic stresses as regulators and substrates through post‐translational phosphorylation.

Modulation of the hippo-YAP pathway by cyclic stretch in rat type 2 alveolar epithelial cells-a proof-of-concept study.

Background: Mechanical ventilation (MV) is a life supporting therapy but may also cause lung damage. This phenomenon is known as ventilator-induced lung injury (VILI). A potential pathomechanisms of ventilator-induced lung injury may be the stretch-induced production and release of cytokines and pro-inflammatory molecules from the alveolar epithelium. Yes-associated protein (YAP) might be regulated by mechanical forces and involved in the inflammation cascade. However, its role in stretch-induced damage of alveolar cells remains poorly understood. In this study, we explored the role of YAP in the response of alveolar epithelial type II cells (AEC II) to elevated cyclic stretch in vitro. We hypothesize that Yes-associated protein activates its downstream targets and regulates the interleukin-6 (IL-6) expression in response to 30% cyclic stretch in AEC II. Methods: The rat lung L2 cell line was exposed to 30% cyclic equibiaxial stretch for 1 or 4h. Non-stretched conditions served as controls. The cytoskeleton remodeling and cell junction integrity were evaluated by F-actin and Pan-cadherin immunofluorescence, respectively. The gene expression and protein levels of IL-6, Yes-associated protein, Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1), and connective tissue growth factor (CTGF/CCN2) were studied by real-time polymerase chain reaction (RT-qPCR) and Western blot, respectively. Verteporfin (VP) was used to inhibit Yes-associated protein activation. The effects of 30% cyclic stretch were assessed by two-way ANOVA. Statistical significance as accepted at p < 0.05. Results: Cyclic stretch of 30% induced YAP nuclear accumulation, activated the transcription of Yes-associated protein downstream targets Cyr61/CCN1 and CTGF/CCN2 and elevated IL-6 expression in AEC II after 1hour, compared to static control. VP (2µM) inhibited Yes-associated protein activation in response to 30% cyclic stretch and reduced IL-6 protein levels. Conclusion: In rat lung L2 AEC II, 30% cyclic stretch activated YAP, and its downstream targets Cyr61/CCN1 and CTGF/CCN2 and proinflammatory IL-6 expression. Target activation was blocked by a Yes-associated protein inhibitor. This novel YAP-dependent pathway could be involved in stretch-induced damage of alveolar cells.

Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane

The γ-aminobutyric acid type B (GABAB) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABAB receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABAB receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments. We further show that GABAB receptors are located together with filamin A in small nanodomains in hippocampal neurons. These interactions are mediated by the first intracellular loop of the GABAB1 subunit and modulate the kinetics of Gαi protein activation in response to GABA stimulation.

Impact of cancer-associated mutations in CC chemokine receptor 2 on receptor function and antagonism

CC chemokine receptor 2 (CCR2), a G protein-coupled receptor, plays a role in many cancer-related processes such as metastasis formation and immunosuppression. Since ∼ 20 % of human cancers contain mutations in G protein-coupled receptors, ten cancer-associated CCR2 mutants obtained from the Genome Data Commons were investigated for their effect on receptor functionality and antagonist binding. Mutations were selected based on either their vicinity to CCR2′s orthosteric or allosteric binding sites or their presence in conserved amino acid motifs. One of the mutant receptors, namely S101P2.63 with a mutation near the orthosteric binding site, did not express on the cell surface. All other studied mutants showed a decrease in or a lack of G protein activation in response to the main endogenous CCR2 ligand CCL2, but no change in potency was observed. Furthermore, INCB3344 and LUF7482 were chosen as representative orthosteric and allosteric antagonists, respectively. No change in potency was observed in a functional assay, but mutations located at F1163.28 impacted orthosteric antagonist binding significantly, while allosteric antagonist binding was abolished for L134Q3.46 and D137N3.49 mutants. As CC chemokine receptor 2 is an attractive drug target in cancer, the negative effect of these mutations on receptor functionality and drugability should be considered in the drug discovery process.

Designing a Novel CRISPR/Cas9 Endogenous Tag of the Amph-1 Gene for Treatment Research Purposes

Amphiphysin 1, or amph-1, is a c. elegans ortholog of the genes amphiphysin (AMPH) and bridging integrator 1 (BIN 1) present in humans. Amph-1 is located primarily in the endomembrane system of the cell. Its protein count is highly affected by diseases under dementia and carcinoma. To gain further understanding of amph-1 protein activity in response to such diseases, this research centers around the creation of a CRISPR/Cas9 design plan to inject modified plasmids into c. elegans eggs and produce specimens available for endogenous activity research. An N20 sequence and 5’ and 3’ homology arms were located near the N-terminus, where a bioluminescent protein tag would be inserted. A plasmid dedicated to synthesizing the Cas9 protein and a second plasmid carrying the genetic code for bioluminescent protein are then modified using amph-1 genetic code as a base template. Following silent mutation edits to the N20 sequence, this plasmid design has potential to be developed in labs for depper research into the amph-1 gene behavior.

Regulation and functional consequences of mGlu4 RNA editing.

Metabotropic glutamate receptor 4 (mGlu4) is one of eight mGlu receptors within the Class C G protein-coupled receptor superfamily. mGlu4 is primarily localized to the presynaptic membrane of neurons where it functions as an auto and heteroreceptor controlling synaptic release of neurotransmitter. mGlu4 is implicated in numerous disorders and is a promising drug target; however, more remains to be understood about its regulation and pharmacology. Using high-throughput sequencing, we have validated and quantified an adenosine-to-inosine (A-to-I) RNA editing event that converts glutamine 124 to arginine in mGlu4; additionally, we have identified a rare but novel K129R site. Using an in vitro editing assay, we then validated the pre-mRNA duplex that allows for editing by ADAR enzymes and predicted its conservation across the mammalian species. Structural modeling of the mGlu4 protein predicts the Q124R substitution to occur in the B helix of the receptor that is critical for receptor dimerization and activation. Interestingly, editing of a receptor homodimer does not disrupt G protein activation in response to the endogenous agonist, glutamate. Using an assay designed to specifically measure heterodimer populations at the surface, however, we found that Q124R substitution decreased the propensity of mGlu4 to heterodimerize with mGlu2 and mGlu7. Our study is the first to extensively describe the extent and regulatory factors of RNA editing of mGlu4 mRNA transcripts. In addition, we have proposed a novel functional consequence of this editing event that provides insights regarding its effects in vivo and expands the regulatory capacity for mGlu receptors.

Optical Control of Cytokine Signaling via Bioinspired, Polymer-Induced Latency.

Cytokine signaling is challenging to study and therapeutically exploit as the effects of these proteins are often pleiotropic. A subset of cytokines can, however, achieve signal specificity via association with latency-inducing proteins, which cage the cytokine until disrupted by discreet biological stimuli. Inspired by this precision, here, we describe a strategy for synthetic induction of cytokine latency via modification with photolabile polymers that mimic latency while attached then restore protein activity in response to light, thus controlling the magnitude, duration, and location of cytokine signals. We characterize the high dynamic range of cytokine activity modulation and find that polymer-induced latency, alone, can prolong in vivo circulation and bias receptor subunit binding. We further show that protein derepression can be achieved with a near single-cell resolution and demonstrate the feasibility of transcutaneous photoactivation. Future extensions of this approach could enable multicolor, optical reprogramming of cytokine signaling networks and more precise immunotherapies.

Photo-regulated control of cytokine signaling via bioinspired, polymer-induced latency

Abstract Cytokine signaling is challenging to study or therapeutically modulate as the effects of these protein are often pleotropic and context-dependent. One mechanism by which cytokines with diverse effects can, however, transmit cell type-specific information is via expression in an inactive, or latent, form in which the protein is sterically shielded by a binding partner that is later displaced or degraded (e.g. TGF-β1 association with GARP or LAP). Inspired by this precision, we hypothesized that photo-reversible chemical modification could impart orthogonal specificity to cytokines not expressed in a latent state, such as IL-2, IL-15, or IL-12. Here, we describe a strategy whereby cytokines are modified with photo-labile polymers that mimic the induction of latency while appended, then de-shield to recover protein activity in response to monochromatic light exposure, thus controlling the magnitude, duration, and location of cytokine signals. Using this strategy, we show that latent IL-2 and IL-15 activity can be photo-modulated as much as 103-fold and that polymer-induced IL-2 latency, alone, can bias affinity away from immunosuppressive CD25 binding. We further show that protein de-repression can be achieved with spatial resolution approaching that of a single immune cell and demonstrate the feasibility of transcutaneous IL-2 photoactivation using engineered tissue phantoms. We find that such modifications also prolong the circulation of IL-12 in vivo, thus obviating the need for frequent, high dosing in therapeutic settings. Future extensions of this approach could enable multicolor, optical reprogramming of cytokine signaling networks and lead to immunotherapies that are more tissue-specific and patient-personalized.

Enzyme-Instructed Activation of Pro-protein Therapeutics In Vivo.

The selective and temporal control of protein activity in living cells provides a powerful tool to manipulate cellular function and to develop pro-protein therapeutics (PPT) for targeted therapy. In this work, we reported a facile but general chemical approach to design PPT by modulating protein activity in response to endogenous enzyme of disease cells, and its potential for targeted cancer therapy. We demonstrated that the chemical modification of a protein with quinone propionic acid (QPN), a ligand that could be reduced by tumor-cell-specific NAD(P)H dehydrogenase [quinone] 1 (NQO1), was reversible in the presence of NQO1. Importantly, the QPN-modified cytochrome c (Cyt c-QPN) and ribonuclease A (RNase A-QPN) showed NQO1-regulated protein activity in a highly selective manner. Furthermore, the intracellular delivery of RNase A-QPN using a novel type of lipid-based nanoparticles, and subsequent protein activation by cellular NQO1, selectively inhibit cancer cell growth in vitro and effectively suppress tumor growth in vivo. We believe that our approach increases the number of potentially useful chemical tools for reversibly controlling the structure and function of protein using a disease-cell-specific enzyme, opening opportunities in the study of dynamic biological processes and developing precise protein therapeutics.

Possible contribution of erythrocytes to the purinergic regulation of tissue oxygen delivery

ATP release occurs in virtually all cell types and tissues. It is considered to be a key component of a ubiquitous, evolutionary ancient cell-to-cell communication system. The regulated release of ATP is also believed to be a part of a mechanism which facilitates matching tissue oxygen supply with demand. In this paper, ATP signaling is reviewed, regulation of tissue oxygen supply is outlined, and a concept attributing a role in this process to erythrocytes releasing ATP is discussed. Oxygen saturation of hemoglobin in erythrocytes traveling through a tissue reflects the level of oxygen utilization of that tissue. Therefore, erythrocytes can serve as local oxygen sensors. In the proposed mechanism, upon hemoglobin deoxygenation, Gi protein in the erythrocyte plasma membrane is activated. The activation of Gi protein initiates a cAMP-dependent pathway, leading to ATP efflux through pannexin channels. Extracellular ATP triggers vasodilatation via P2Y receptors on the surface of vascular endothelial cells, increasing blood flow in tissue regions of elevated oxygen consumption. Despite the abundance of compelling evidence in support of this concept, some details remain elusive, in particular, the process of Gi protein activation in response to hemoglobin desaturation. Furthermore, the involvement of cAMP, as well as the final conduit of ATP release from erythrocytes, remains controversial. Finally, the actual physiological relevance of the proposed regulatory mechanism will require further in vivo research.

Mek1Y130C mice recapitulate aspects of human cardio-facio-cutaneous syndrome.

ABSTRACTThe RAS/MAPK signaling pathway is one of the most investigated pathways, owing to its established role in numerous cellular processes and implication in cancer. Germline mutations in genes encoding members of the RAS/MAPK pathway also cause severe developmental syndromes collectively known as RASopathies. These syndromes share overlapping characteristics, including craniofacial dysmorphology, cardiac malformations, cutaneous abnormalities and developmental delay. Cardio-facio-cutaneous syndrome (CFC) is a rare RASopathy associated with mutations in BRAF, KRAS, MEK1 (MAP2K1) and MEK2 (MAP2K2). MEK1 and MEK2 mutations are found in ∼25% of the CFC patients and the MEK1Y130C substitution is the most common one. However, little is known about the origins and mechanisms responsible for the development of CFC. To our knowledge, no mouse model carrying RASopathy-linked Mek1 or Mek2 gene mutations has been reported. To investigate the molecular and developmental consequences of the Mek1Y130C mutation, we generated a mouse line carrying this mutation. Analysis of mice from a Mek1 allelic series revealed that the Mek1Y130C allele expresses both wild-type and Y130C mutant forms of MEK1. However, despite reduced levels of MEK1 protein and the lower abundance of MEK1 Y130C protein than wild type, Mek1Y130C mutants showed increased ERK (MAPK) protein activation in response to growth factors, supporting a role for MEK1 Y130C in hyperactivation of the RAS/MAPK pathway, leading to CFC. Mek1Y130C mutant mice exhibited pulmonary artery stenosis, cranial dysmorphia and neurological anomalies, including increased numbers of GFAP+ astrocytes and Olig2+ oligodendrocytes in regions of the cerebral cortex. These data indicate that the Mek1Y130C mutation recapitulates major aspects of CFC, providing a new animal model to investigate the physiopathology of this RASopathy.This article has an associated First Person interview with the first author of the paper.

Acetylome Analysis Identifies SIRT1 Targets in mRNA-Processing and Chromatin-Remodeling in Mouse Liver.

Lysine acetylation is a post-translational modification found on numerous proteins, a strategy used in cell signaling to change protein activity in response to internal or external cues. Sirtuin 1 (SIRT1) is a central lysine deacetylase involved in a variety of cellular processes including metabolism, apoptosis, and DNA repair. Here we characterize the lysine acetylome in mouse liver, and by using a model of Sirt1 -/-knockout mouse, show that SIRT1 regulates the deacetylation of 70 proteins in the liver in-vivo. Amongst these SIRT1-regulated proteins, we find that four RNA-processing proteins and a chromatin-remodeling protein can be deacetylated by SIRT1 directly in-vitro. The discovery that SIRT1 has a potential role in RNA-processing suggests a new layer of regulation in the variety of functions performed by SIRT1.

Altered G Protein Coupling in Olfactory Neuroepithelial Cells From Patients With Schizophrenia

Increasing evidence suggests that olfactory dysfunction is an endophenotype of schizophrenia, and thus the olfactory system can be studied both in relation to this sensory dysfunction and also as a means of examining pathophysiologic mechanisms of schizophrenia. In this study, we examined human olfactory neuroepithelial (ON) biopsy tissues and their in vitro culture cells for ligand-induced guanine nucleotide-binding protein (G protein) activation and downstream signaling. We assessed the binding of a nonhydrolyzable GTP analogue [(35)S]GTPγS binding to specific G protein subtypes in response to odorants, dopamine, or serotonin in ON cell membranes from matched schizophrenia-control subjects. In response to odorant mixtures, we found decreased [(35)S]GTPγS binding to Gαs/olf in schizophrenia patients. These changes were not mediated by mRNA expression of key molecules of G protein coupling, including adenylate cyclase III (ACIII), protein kinase A (PKA), protein kinase Cγ (PKCγ), or Gαs or Gαolf in ON cells or ON biopsy tissues. In contrast, dopamine (DA)- and serotonin (5HT)-induced S(35)-GTPγS binding to Gαs/olf and Gαq/11 were significantly increased in schizophrenia cases, while these parameters were strikingly reduced by in vitro treatment with antipsychotics. Patients with schizophrenia exhibit increases in electrolfactogram (EOG) recordings, suggesting enhanced odorant-induced activation. Our results of decreased odorant-induced G protein activation may point further downstream for underlying mechanisms for increased EOG measures. Increased G protein activation in response to DA and 5HT may suggest increased postreceptor DA or 5HT signaling as an additional mechanism of dopaminergic or serotonergic dysregulation in schizophrenia.

Interfacing synthetic DNA logic operations with protein outputs.

DNA logic gates are devices composed entirely of DNA that perform Boolean logic operations on one or more oligonucleotide inputs. Typical outputs of DNA logic gates are oligonucleotides or fluorescent signals. Direct activation of protein function has not been engineered as an output of a DNA-based computational circuit. Explicit control of protein activation enables the immediate triggering of enzyme function and could yield DNA computation outputs that are otherwise difficult to generate. By using zinc-finger proteins, AND, OR, and NOR logic gates were created that respond to short oligonucleotide inputs and lead to the activation or deactivation of a split-luciferase enzyme. The gate designs are simple and modular, thus enabling integration with larger multigate circuits, and the modular structure gives flexibility in the choice of protein output. The gates were also modified with translator circuits to provide protein activation in response to microRNA inputs as potential cellular cancer markers.

Interfacing Synthetic DNA Logic Operations with Protein Outputs

AbstractDNA logic gates are devices composed entirely of DNA that perform Boolean logic operations on one or more oligonucleotide inputs. Typical outputs of DNA logic gates are oligonucleotides or fluorescent signals. Direct activation of protein function has not been engineered as an output of a DNA‐based computational circuit. Explicit control of protein activation enables the immediate triggering of enzyme function and could yield DNA computation outputs that are otherwise difficult to generate. By using zinc‐finger proteins, AND, OR, and NOR logic gates were created that respond to short oligonucleotide inputs and lead to the activation or deactivation of a split‐luciferase enzyme. The gate designs are simple and modular, thus enabling integration with larger multigate circuits, and the modular structure gives flexibility in the choice of protein output. The gates were also modified with translator circuits to provide protein activation in response to microRNA inputs as potential cellular cancer markers.

Increasing levels of cardiolipin differentially influence packing of phospholipids found in the mitochondrial inner membrane

It is essential to understand the role of cardiolipin (CL) in mitochondrial membrane organization given that changes in CL levels contribute to mitochondrial dysfunction in type II diabetes, ischemia–reperfusion injury, heart failure, breast cancer, and aging. Specifically, there are contradictory data on how CL influences the molecular packing of membrane phospholipids. Therefore, we determined how increasing levels of heart CL impacted molecular packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures found within the mitochondrial inner membrane, using merocyanine (MC540) fluorescence. We broadly categorized lipid vesicles of equal mass as loosely packed, intermediate, and highly packed based on peak MC540 fluorescence intensity. CL had opposite effects on loosely versus highly packed vesicles. Exposure of loosely packed vesicles to increasing levels of CL dose-dependently increased membrane packing. In contrast, increasing amounts of CL in highly packed vesicles decreased the packing in a dose-dependent manner. In vesicles that were categorized as intermediate packing, CL had either no effect or decreased packing at select doses in a dose-independent manner. Altogether, the results aid in resolving some of the discrepant data by demonstrating that CL displays differential effects on membrane packing depending on the composition of the lipid environment. This has implications for mitochondrial protein activity in response to changing CL levels in microdomains of varying composition.

Real-time detection of G protein activation using monomolecular Gγ FRET sensors

G protein-coupled receptors (GPCRs) are involved in many diseases, and are the target of a large percentage of modern drugs. While only a few fluorescence resonance energy transfer (FRET) sensors have been made for real-time detection of GPCR activation, the human genome encodes roughly 950 GPCRs and a need for a broad real-time detection system is needed. In this study, we developed and characterized a new type of G protein sensor based on the Gγ subunit containing an intra-molecular FRET pair to allow detection of real-time G protein activation in multiple cell lines using time-resolved fluorescence spectroscopy. Our Gγ sensors were able to detect G protein activation to aluminum fluoride, a G protein activator, in human embryonic kidney 293 cells (HEK293). Moreover, our sensors were able to couple to the bradykinin B2, parathyroid type 1 and adrenergic 2A GPCRs and detect G protein activation in response to the cognate ligands; bradykinin, parathyroid and noradrenaline hormones, respectively. Furthermore, our sensors also detected ligand-independent G protein activation by fluid shear stress. G protein inhibitors, pertussis toxin and guanosine 5′ [β-thio] diphosphate, inhibited the FRET response to G protein stimulants indicating that the sensor response is specific to G protein activation. These findings suggest that the described Gγ sensors can be used to monitor real-time G protein activation by a variety of G protein stimulants or inhibitors.

Expression and Function of Bile Acid Receptor TGR5 in Gastrointestinal Smooth Muscle

Bile acids, the main active constituents of bile, act, in addition to their role in nutrient absorption, as signaling molecules to exert genomic and non-genomic effects via nuclear receptors and the plasma membrane G protein-coupled receptor TGR5. TGR5 is expressed in epithelial cells and enteric neurons of the gastrointestinal tract and may mediate the effects of bile acids on motility and secretion. However, the mechanisms by which bile acids regulate smooth muscle function are not known. AIM To determine the expression of TGR5 in gastrointestinal smooth muscle and identify their signaling pathways. METHODS TGR5 expression was determined by RT-PCR and western blot. G protein activation in response to TGR5 selective ligand, oleanolic acid (OA) was measured as increase in Gα binding to [35S]GTPγS. OA-induced increase in cAMP was measured by ELISA and muscle relaxation by scanning micrometry. RESULTS TGR5 expression was detected by RT-PCR and western blot in rabbit gastric muscle and human intestinal muscle. OA activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3. Consistent with activation of Gαs, OA increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced muscle contraction. CONCLUSION Smooth muscle cells express membrane receptors (TGR5) for bile acids preferentially coupled to Gs. The receptors mediate stimulation of adenylyl cyclase activity and muscle relaxation.

ARF-dependent regulation of ATM and p53 associated KZNF (Apak) protein activity in response to oncogenic stress

The KRAB-type zinc-finger protein Apak (ATM and p53 associated KZNF protein) specifically suppresses p53-mediated apoptosis. Upon DNA damage, Apak is phosphorylated and inhibited by ATM kinase, resulting in p53 activation. However, how Apak is regulated in response to oncogenic stress remains unknown. Here we show that upon oncogene activation, Apak is inhibited in the tumor suppressor ARF-dependent but ATM-independent manner. Oncogene-induced ARF protein directly interacts with Apak and competes with p53 to bind to Apak, resulting in Apak dissociation from p53. Thus, Apak is differentially regulated in the ARF and ATM-dependent manner in response to oncogenic stress and DNA damage, respectively. Structured summary MINT- 7989670: p53 (uniprotkb: P04637) binds (MI: 0407) to APAK (uniprotkb: Q8TAQ5) by pull down (MI: 0096)MINT- 7989812: HDM2 (uniprotkb: Q00987) physically interacts (MI: 0915) with ARF (uniprotkb: Q8N726-1) by anti bait coimmunoprecipitation (MI: 0006)MINT- 7989603, MINT- 7989626: APAK (uniprotkb: Q8TAQ5) physically interacts (MI: 0915) with ARF (uniprotkb: Q8N726-1) by anti bait coimmunoprecipitation (MI: 0006)MINT- 7989653: ARF (uniprotkb: Q8N726-1) binds (MI: 0407) to APAK (uniprotkb: Q8TAQ5) by pull down (MI: 0096)MINT- 7989686, MINT- 7989705, MINT- 7989747: APAK (uniprotkb: Q8TAQ5) physically interacts (MI: 0915) with ARF (uniprotkb: Q8N726-1) by anti tag coimmunoprecipitation (MI: 0007)MINT- 7989724: APAK (uniprotkb: Q8TAQ5) physically interacts (MI: 0914) with ARF (uniprotkb: Q8N726-1) and p53 (uniprotkb: P04637) by anti tag coimmunoprecipitation (MI: 0007)MINT- 7989635: ARF (uniprotkb: Q8N726-1) and APAK (uniprotkb: Q8TAQ5) colocalize (MI: 0403) by fluorescence microscopy (MI: 0416)MINT- 7989584, MINT- 7989773: APAK (uniprotkb: Q8TAQ5) physically interacts (MI: 0915) with p53 (uniprotkb: P04637) by anti tag coimmunoprecipitation (MI: 0007)