Resonance Energy Transfer Assay Research Articles

Detection of misfolded rhodopsin aggregates in cells by Förster resonance energy transfer.

Rhodopsin is the light receptor in rod photoreceptor cells of the retina that plays a central role in phototransduction and rod photoreceptor cell health. Rhodopsin mutations are the leading known cause of autosomal dominant retinitis pigmentosa, a retinal degenerative disease. A majority of rhodopsin mutations cause misfolding and aggregation of the apoprotein opsin. The nature of aggregates formed by misfolded rhodopsin mutants and the associated cell toxicity is poorly understood. Misfolding rhodopsin mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand rhodopsin aggregation, disease pathogenesis, and evaluate therapeutic strategies. To better understand the aggregation of misfolded rhodopsin mutants, a Förster resonance energy transfer assay has been developed to monitor the aggregation of fluorescently tagged mutant rhodopsins expressed in live cells.

Activating PRKACB somatic mutation in cortisol-producing adenomas

Mutations in the gene encoding the protein kinase A (PKA) catalytic subunit α have been found to be responsible for cortisol-producing adenomas (CPAs). In this study, we identified by whole-exome sequencing the somatic mutation p.S54L in the PRKACB gene, encoding the catalytic subunit β (Cβ) of PKA, in a CPA from a patient with severe Cushing syndrome. Bioluminescence resonance energy transfer and surface plasmon resonance assays revealed that the mutation hampers formation of type I holoenzymes and that these holoenzymes were highly sensitive to cAMP. PKA activity, measured both in cell lysates and with recombinant proteins, based on phosphorylation of a synthetic substrate, was higher under basal conditions for the mutant enzyme compared with the WT, while maximal activity was lower. These data suggest that at baseline the PRKACB p.S54L mutant drove the adenoma cells to higher cAMP signaling activity, probably contributing to their autonomous growth. Although the role of PRKACB in tumorigenesis has been suggested, we demonstrated for the first time to our knowledge that a PRKACB mutation can lead to an adrenal tumor. Moreover, this observation describes another mechanism of PKA pathway activation in CPAs and highlights the particular role of residue Ser54 for the function of PKA.

7-O-Methylwogonin from Scutellaria baicalensis Disturbs Mitotic Progression by Inhibiting Plk1 Activity in Hep3B Cells.

Polo-like kinase 1, a mitotic Ser/Thr kinase, has emerged as a molecular target for the development of anticancer drugs. In this study, we found that polo-like kinase 1 activity was inhibited by 7-O-methylwogonin and related flavones, including baicalein, dihydrobaicalein, and viscidulin II, isolated from Scutellaria baicalensis. Although dihydrobaicalein exhibited the highest polo-like kinase 1 inhibitory activity among the four compounds, it also inhibited other kinases, such as vaccinia-related kinase 2 and polo-like kinase 2. Baicalein and viscidulin II also showed low selectivity to polo-like kinase 1 since they inhibited polo-like kinase 3 and polo-like kinase 2, respectively. However, 7-O-methylwogonin exhibited selective polo-like kinase 1 inhibitory activity, as evidenced from in vitro kinase assays based on fluorescence resonance energy transfer assays and ADP-Glo kinase assays. In addition, examination of mitotic morphology and immunostaining using specific antibodies for the mitotic markers, p-histone H3 and mitotic protein monoclonal 2, in Hep3B cells showed that 7-O-methylwogonin treatment increased mitotic cell populations due to inhibition of mitotic progression as a result of polo-like kinase 1 inhibition. The pattern of 7-O-methylwogonin-induced mitotic arrest was similar to that of BI 2536, a specific polo-like kinase 1 inhibitor. Thus, it was suggested that 7-O-methylwogonin disturbed mitotic progression by inhibiting polo-like kinase 1 activity. These data suggest that 7-O-methylwogonin, a polo-like kinase 1 inhibitor, may be a useful anticancer agent because of its polo-like kinase 1 selectivity and effectiveness.

The porcine odorant-binding protein as molecular probe for benzene detection.

In recent years, air pollution has been a subject of great scientific and public interests for the strong impact on human health. Air pollution is due to the presence in the atmosphere of polluting substances, such as carbon monoxide, sulfur and nitrogen oxides, particulates and volatile organic compounds (VOCs), derived predominantly from various combustion processes. Benzene is a VOC belonging to group-I carcinogens with a toxicity widely demonstrated. The emission limit values and the daily exposure time to benzene (TLV-TWA) are 5μg/m3 (0.00157 ppm) and 1.6mg/m3 (0.5 ppm), respectively. Currently, expensive and time-consuming analytical methods are used for detection of benzene. These methods require to perform a few preliminary steps such as sampling, and matrices pre-treatments. In addition, it is also needed the support of specialized personnel. Recently, single-walled carbon nanotube (SWNTs) gas sensors with a limit detection (LOD) of 20 ppm were developed for benzene detection. Other innovative bioassay, called bio-report systems, were proposed. They use a whole cell (Pseudomona putida or Escherichia coli) as molecular recognition element and exhibit a LOD of about 10 μM. Here, we report on the design of a highly sensitive fluorescence assay for monitoring atmospheric level of benzene. For this purpose, we used as molecular recognition element the porcine odorant-binding protein (pOBP). 1-Aminoanthracene was selected as extrinsic fluorescence probe for designing a competitive fluorescence resonance energy transfer (FRET) assay for benzene detection. The detection limit of our assay was 3.9μg/m3, a value lower than the actual emission limit value of benzene as regulated by European law.

Identification of amentoflavone as a potent highly selective PARP-1 inhibitor and its potentiation on carboplatin in human non-small cell lung cancer

Nuclear protein poly (ADP-ribose) polymerase-1 (PARP-1) is a key enzyme in the repair of DNA and is a promising target in the development of chemosensitizers. This study first investigated the inhibitory effects of amentoflavone (AMF) and its derivatives on PARP-1 and the potentiation of AMF on carboplatin (CBP) in non-small cell lung cancer (NSCLC). This study aims to evaluate the inhibitory effect of AMF against PARP-1 and its potentiation on CBP in lung cancer both in vitro and in vivo. The inhibitory effect of AMF on PARP-1 was investigated using molecular docking and cell-free model of PARP-1 assay. Its potentiation on CBP in lung cancer was also evaluated. Fluorescence resonance energy transfer assay was used to detect the inhibitory effects of AMF and its analogues on PARP-1. Molecular docking was employed to predict the binding mode of AMF and PARP-1. MTT assay, isobologram analysis, Hoechst staining, and Annexin V-PI double staining were used to confirm the potentiation of AMF on CBP in vitro. siRNA (PARP-1)-A549 cells were used to reveal the action target of AMF. Western blot analysis, immunohistochemistry, and Tunnel assay were employed to evaluate the potentiation of AMF on CBP in A549 xenograft mice. AMF and its analogues exerted excellent inhibitory effects on PARP-1 with IC50 values ranging from 0.198 μM to 0.409 μM. Docking experiment showed that AMF can stably bind to PARP-1 with a comparable binding energy to olaparib. AMF can decrease the expression of PAR induced by H2O2in vitro. AMF synergistically increased the CBP anti-proliferative effect in A549. However, its potentiation nearly disappeared when the cells were transfected with siRNAs against PARP-1. Oral administration of AMF (100 mg/kg), combined with CBP, remarkably inhibited A549 tumor growth and ki67 expression, and increased apoptosis compared with CBP-alone group. All results suggest that AMF can be a potential PARP-1 inhibitor and a candidate adjuvant agent to boost the anticancer effect of CBP in NSCLC.

Nonhormonal selective estrogen receptor modulator 1-(2-[4-{(3R,4S)-7-Methoxy-2, 2-dimethyl-3-phenyl-chroman-4yl}phenoxy]ethyl)pyrrolidine hydrochloride (ormeloxifene hydrochloride) for the treatment of breast cancer.

Breast cancer is the most common type of diagnosed cancers in women, difficult to treat, and has received international attention because of its aggressive nature and inherent drug resistance mechanisms. Development of a better selective estrogen receptor modulator with good therapeutic profile and less toxicity is very crucial in this scenario. This study was undertaken to evaluate and compare the in vitro and in vivo antitumor activities of ormeloxifene with other clinically used breast cancer drugs. Cytotoxic activity of ormeloxifene was compared with standard drugs, 4-hydroxytamoxifene and Adriamycin. Ormeloxifene (50 μM) concentration showed cytotoxicity of 75% and 82% in MDAMB-231 and 24% and 80% in MCF-7 cells, respectively, after 72 and 144 hr of incubation as displayed by cell viability assay. The same concentration of ormeloxifene was shown to exert 74% caspase-7 activation in MCF-7 cells after 24 hr of incubation by fluorescence resonance energy transfer assay. Cell cycle analysis proved that there was an increase in sub-G1 peak to 64.4% and 33.9% in MDAMB-231 and MCF-7 cells, respectively, after treatment using ormeloxifene (50 μM) for 48 hr. The nonobese diabetic-severe combined immunodeficiency mice bearing tumor xenografts of triple negative MDAMB-231 cells treated with ormeloxifene (3 mg/kg bw) showed significant regression in relative tumor volume compared to control. From the results obtained and as evidenced from prior literature, ormeloxifene in addition to contraceptive use, can be repositioned for the development of an efficacious anticancer drug. These data present the preclinical part of a well concerted effort to place ormeloxifene into further clinical trials.

Abstract P256: Development of an Automated High Throughput Screen for GRK4 as Potential Novel Therapeutics for Treatment of Hypertension and/or Salt Sensitivity

G protein coupled kinase type 4 (GRK4) reduces renal sodium excretion by deactivating the renal dopamine type 1 receptor (D 1 R) through serine phosphorylation. At least 3 polymorphisms in GRK4 have been shown to be associated with the expression of hypertension and/or salt sensitivity in humans and in mice. GRK4 is an ideal therapeutic target for treating hypertension since putting human GRK4 variants in mice results in hypertension which can be reversed by reducing the expression of GRK4. Inhibitors of GRK4 could serve as potent and selective agents to promote sodium excretion in salt sensitive and/or hypertensive individuals. Therefore, we developed an automated high throughput homogeneous time resolved fluorescent resonance energy transfer assay (TR-FRET) to identify small molecule inhibitors of GRK4 isoenzymes. The assay relies on the close proximity (90 A) transfer of 337 nm energy from a europium anti-phosphoserine antibody (Eu-pSer) to an allophycocyanin labeled streptavidin acceptor (streptavidin-APC), producing FRET at 665 nm. The assay was optimized through serial dilutions of streptavidin-APC, Eu-pSer, GRK4, and a peptide consisting of serine sites in the D 1 R, serving as a substrate. To validate the assay and determine its suitability for a large scale high throughput screen, three inhibitors, GRKA, GRKB, and GRKC were selected and concentration response assays were performed to determine IC50 values and variability analysis. The IC50 and (R 2 ) values of GRKA, GRKB, and GRKC were 4.99 μM (0.99), 26.52 μM (0.99), and 179.8 μM (0.82), respectively. GRKA is the most efficient GRK4 inhibitor, while GRKC is the least. This assay will be used to test congeners of compounds A, B, and C to identify more selective candidates, as well as for screening available small molecule compound libraries.

Mapping the Interface of a GPCR Dimer: A Structural Model of the A2A Adenosine and D2 Dopamine Receptor Heteromer.

The A2A adenosine (A2AR) and D2 dopamine (D2R) receptors form oligomers in the cell membrane and allosteric interactions across the A2AR–D2R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A2AR–D2R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A2AR–D2R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A2AR–D2R receptor–receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A2AR blocked heterodimer interactions and disrupted the allosteric effect of A2AR activation on D2R agonist binding. Protein–protein docking was used to construct a model of the A2AR–D2R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A2AR–D2R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A2AR–D2R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.

Ophthalmic Nonsteroidal Anti-Inflammatory Drugs as a Therapy for Corneal Dystrophies Caused by SLC4A11 Mutation.

SLC4A11 is a plasma membrane protein of corneal endothelial cells. Some mutations of the SLC4A11 gene result in SLC4A11 protein misfolding and failure to mature to the plasma membrane. This gives rise to some cases of Fuchs' endothelial corneal dystrophy (FECD) and congenital hereditary endothelial dystrophy (CHED). We screened ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs) for their ability to correct SLC4A11 folding defects. Five ophthalmic NSAIDs were tested for their therapeutic potential in some genetic corneal dystrophy patients. HEK293 cells expressing CHED and FECD-causing SLC4A11 mutants were grown on 96-well dishes in the absence or presence of NSAIDs. Ability of NSAIDs to correct mutant SLC4A11 cell-surface trafficking was assessed with a bioluminescence resonance energy transfer (BRET) assay and by confocal microscopy. The ability of mutant SLC4A11-expressing cells to mediate water flux (SLC4A11 mediates water flux across the corneal endothelial cell basolateral membrane as part of the endothelial water pump) was measured upon treatment with ophthalmic NSAIDs. BRET-assays revealed significant rescue of SLC4A11 mutants to the cell surface by 4 of 5 NSAIDs tested. The NSAIDs, diclofenac and nepafenac, were effective in moving endoplasmic reticulum-retained missense mutant SLC4A11 to the cell surface, as measured by confocal immunofluorescence. Among intracellular-retained SLC4A11 mutants, 20 of 30 had significant restoration of cell surface abundance upon treatment with diclofenac. Diclofenac restored mutant SLC4A11 water flux activity to the level of wild-type SLC4A11 in some cases. These results encourage testing diclofenac eye drops as a treatment for corneal dystrophy in patients whose disease is caused by some SLC4A11 missense mutations.

In Vitro Pharmacological Characterization of Buprenorphine, Samidorphan, and Combinations Being Developed as an Adjunctive Treatment of Major Depressive Disorder.

A combination of buprenorphine (BUP) and samidorphan (SAM) at a 1:1 (mg/mg) fixed-ratio dose is being investigated as an adjunctive treatment of major depressive disorder (BUP/SAM, ALKS 5461). Both [3H]BUP and [3H]SAM bound to the μ-, κ-, and δ-opioid receptors (MOR, KOR, and DOR, respectively) with Kd values of 3 nM or less. [3H]BUP dissociated from the MOR more slowly than [3H]SAM did. In the [35S]GTPγS assay, BUP was a partial agonist at the MOR, KOR, and DOR. SAM was an antagonist at the MOR and a partial agonist at the KOR and DOR. The pharmacology of the combination of SAM and BUP was characterized at ratios like the molar ratios of both compounds at steady state in humans. In all assessments, SAM reduced the efficacy of BUP at the MOR without altering its potency. At the KOR, SAM had no significant effect on the activity of BUP. In bioluminescent resonance energy transfer assays, SAM, naltrexone, and naloxone were partial agonists when the MOR was coupled to the Gα oB and Gα z, and were antagonists when coupled to Gα i At the KOR, SAM was a partial agonist activating Gα oA and Gα oB and a full agonist in stimulating Gα z SAM inhibited BUP's recruitment of β-arrestin to the MOR, suggesting an attenuation of BUP's efficacy in activating G proteins correlated with an inhibition of β-arrestin recruitment. The collective data suggest that SAM attenuates the efficacy of BUP under all conditions tested at the MOR and DOR but had little effect on BUP activity at the KOR.

Abstract LB-327: Identification of novel sodium iodide symporter (NIS) interactors which modulate iodide uptake

Abstract Ablative radioiodide therapy is an effective treatment for thyroid cancer, but around a quarter of patients do not accumulate therapeutically-sufficient doses of radioiodide due to decreased expression and/or plasma membrane (PM) localization of the sodium iodide symporter (NIS). Putative use of radioiodide therapy in breast cancer is hampered by low levels of NIS membrane localisation. Currently, the regulation of NIS trafficking is ill-defined. Mass spectrometry was performed on proteins co-immunoprecipitating with lentivirally expressed NIS in whole cell and plasma membrane extracts. NIS activity was significantly altered by ADP-ribosylation factor 4 (ARF4) and valosin containing protein (VCP) in TPC1 thyroid and MDA-MB-231 breast cancer cells lentivirally-expressing NIS. ARF4 downregulation markedly decreased radioiodide uptake, and VCP downregulation increased radioiodide uptake in thyroid and breast cells. Transient overexpression of these genes significantly reversed siRNA effects on NIS function. Co-immunoprecipitation assays confirmed NIS interacts with ARF4 and VCP in vitro, and proximity ligation assays revealed the subcellular sites of interaction. Critically, human primary thyroid culture experiments confirmed that ARF4 and VCP regulate endogenous NIS function in vivo. In addressing the mechanisms of NIS trafficking we identified a C-terminal VXPX motif of NIS which is required for Arf4 regulation, whilst TIRF microscopy revealed coincident Arf4 and NIS localization to shared PM vesicles. Pharmacological inhibitor studies identified that the VCP inhibitors Eeyarastatin-1 and NMS-873 overcome VCP inhibition of NIS function. VCP, which is central to ER-associated Degradation (ERAD), selectively targeted a dimeric form of NIS, which we now identify through co-immunoprecipitation, 3D modelling and Förster resonance energy transfer (FRET) assays. TCGA data analysis of 58 matched papillary thyroid cancers revealed that Arf4 was significantly repressed and VCP highly upregulated in thyroid cancer, providing a putative explanation for repressed NIS function. Our data suggest a possible model for NIS trafficking: NIS dimerises in the ER, a process monitored by VCP and the ERAD pathway, with correctly processed and folded NIS being trafficked to the PM via C-terminal interaction with Arf4. We thus now identify two new potential therapeutic targets for enhancing radioiodide uptake in patients with radioiodide-refractory thyroid cancer. Citation Format: Alice Fletcher, Rebecca Thompson, Martin Read, Andrew Turnell, Vicki Smith, Chris J. McCabe. Identification of novel sodium iodide symporter (NIS) interactors which modulate iodide uptake [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-327.

Abstract 2662: Indenoisoquinoline topoisomerase I inhibitors target the DNA G-quadruplex formed in the c-Myc promoter and cause MYC downregulation

Abstract The inhibition of DNA topoisomerase I (Top1) has proven to be a successful approach toward the design of anticancer agents. Indenoisoquinoline derivatives have been designed and developed as Top1 inhibitors and can overcome some shortcomings associated with the camptothecin Top1 inhibitors, resulting in three indenoisoquinoline compounds, indotecan (LMP 400), indimitecan (LMP776), and LMP744 entering Phase I clinical trials at the National Cancer Institute. However, some active indenoisoquinolines did not show potent Top1 inhibition, suggesting a separate mechanism of action. c-Myc is one of the most commonly deregulated genes in human cancers; a DNA G-quadruplex (G4) formed in the proximal promoter region of c-Myc has been found to function as a transcriptional silencer and be amenable to small molecule targeting. As indenoisoquinolines share structure similarity to our c-Myc G-quadruplex (MycG4)-interactive compounds, we hypothesized that indenoisoquinolines could stabilize MycG4 and downregulate MYC transcription. Using a fluorescence resonance energy transfer (FRET) assay, we examined the MycG4 stabilization activity of 56 indenoisoquinoline compounds that we previously developed as Top1 inhibitors. Notably, 26 of the 56 compounds decreased the fluorescence intensity by at least 50%, and the 10 most potent compounds reduced fluorescence intensities by over 70%, indicating that these indenoisoquinolines are effective MycG4 stabilizers. The identified compounds were further characterized by fluorescence anisotropy, circular dichroism, and nuclear magnetic resonance for MycG4 interactions, followed by western blot assays for MYC downregulation. Significantly, the compounds with good MycG4 binding activity and affinity exhibited significant dose-dependent reduction of MYC expression, indicating that targeting the c-Myc promoter G-quadruplex is a likely mechanism of action for these active anticancer compounds. Furthermore, some active indenoisoquinolines exhibited both MYC downregulation and Top1 inhibition, suggesting that dual targeting c-Myc G-quadruplex and Top1 may represent a novel mechanism of action for anticancer drug design. Citation Format: Kaibo Wang, Mohamed S. A. Elsayed, Guanhui Wu, Mark Cushman, Danzhou Yang. Indenoisoquinoline topoisomerase I inhibitors target the DNA G-quadruplex formed in the c-Myc promoter and cause MYC downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2662.

Abstract 2863: Dissecting the structural basis for inhibitors of RNA-binding proteins

Abstract RNA-binding proteins (RBPs) are key regulators of cellular functions, especially in post-transcriptional regulations. Dysregulation of RBPs is implicated in many diseases including cancer. One of the RBPs that is overexpressed in a variety of human cancer is Musashi-2 (MSI2). Elevated MSI2 expression is associated with ectopic oncogenic pathways, including but not limited to NUMB/Notch, PTEN/mTOR, TGF-β/SMAD3, making MSI2 a promising therapeutic target for cancer. In the case of NUMB, MSI2 binds to and negatively regulates translation of NUMB, a negative regulator of Notch signaling. Protein structure is critical for drug discovery and structure-based rational design. However, so far there is no structure available for MSI2 protein. Recently, using nuclear magnetic resonance (NMR) we solved the first solution structure of MSI2 RNA-recognition Motif 1 (MSI2-RRM1) that forms the major long narrow RNA-binding pocket. Using a fluorescence polarization (FP) chemical libraries screening, we identified several groups of hit compounds that disrupt the binding of MSI2-NUMB RNA potently. These compounds induced apoptosis, inhibited cancer cell proliferation, invasion and metastasis in multiple tumor models tested in vitro and in vivo. They also interfered with cancer stem cell functions with reduced tumorsphere formation. Several lead compounds showed promising efficacy in animal tumor models of human breast, prostate and colon cancer. To investigate the specificity of the compounds towards other RBPs, the compounds were tested in biophysical binding assays such as FP, time resolved Fluorescence Resonance Energy Transfer assay (TR-FRET), NMR spectroscopy and cell-based assays. We compared the affinity of these MSI2 inhibitors towards other RBPs such as MSI1 and HuR. Using docking and molecular dynamics simulation, we analyzed the structure-activity relationship of these inhibitors towards different RBP targets. This work adds significant information to the structure of MSI2-RRM1 and the structural basis for designing more potent and specific inhibitors of RBPs. Citation Format: Lan Lan, Minli Xing, Xiaoqing Wu, Philip Gao, Justin T. Douglas, Yu Zhan, Gulhumay Gardashova, Jiajun Liu, Robert P. Hanzlik, Jeffrey Aubé, Kristi L. Neufeld, Berl R. Oakley, Roberto N. De Guzman, Liang Xu. Dissecting the structural basis for inhibitors of RNA-binding proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2863.

Animal Hen1 2'-O-methyltransferases as tools for 3'-terminal functionalization and labelling of single-stranded RNAs.

S-adenosyl-L-methionine-dependent 2′-O-methylati-on of the 3′-terminal nucleotide plays important roles in biogenesis of eukaryotic small non-coding RNAs, such as siRNAs, miRNAs and Piwi-interacting RNAs (piRNAs). Here we demonstrate that, in contrast to Mg2+/Mn2+-dependent plant and bacterial homologues, the Drosophila DmHen1 and human HsHEN1 piRNA methyltransferases require cobalt cations for their enzymatic activity in vitro. We also show for the first time the capacity of the animal Hen1 to catalyse the transfer of a variety of extended chemical groups from synthetic analogues of the AdoMet cofactor onto a wide range (22–80 nt) of single-stranded RNAs permitting their 3′-terminal functionalization and labelling. Moreover, we provide evidence that deletion of a small C-terminal region of the DmHen1 protein further increases its modification efficiency and abolishes a modest 3′-terminal nucleotide bias observed for the full-length protein. Finally, we show that fluorophore-tagged ssRNA molecules are successfully detected in fluorescence resonance energy transfer assays both individually and in a total RNA mixture. The presented DmHen1-assisted RNA labelling provides a solid basis for developing novel chemo-enzymatic approaches for in vitro studies and in vivo monitoring of single-stranded RNA pools.

Multivalent Cation-Bridged PI(4,5)P2 Clusters Form at Very Low Concentrations

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2), is a key component of the inner leaflet of the plasma membrane in eukaryotic cells. In model membranes, PIP2 has been reported to form clusters, but whether these locally different conditions could give rise to distinct pools of unclustered and clustered PIP2 is unclear. By use of both fluorescence self-quenching and Förster resonance energy transfer assays, we have discovered that PIP2 self-associates at remarkably low concentrations starting below 0.05 mol% of total lipids. Formation of these clusters was dependent on physiological divalent metal ions, such as Ca2+, Mg2+, Zn2+, or trivalent ions Fe3+ and Al3+. Formation of PIP2 clusters was also headgroup-specific, being largely independent of the type of acyl chain. The similarly labeled phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol exhibited no such clustering. However, six phosphoinositide species coclustered with PIP2. The degree of PIP2 cation clustering was significantly influenced by the composition of the surrounding lipids, with cholesterol and phosphatidylinositol enhancing this behavior. We propose that PIP2 cation-bridged cluster formation, which might be similar to micelle formation, can be used as a physical model for what could be distinct pools of PIP2 in biological membranes. To our knowledge, this study provides the first evidence of PIP2 forming clusters at such low concentrations. The property of PIP2 to form such clusters at such extremely low concentrations in model membranes reveals, to our knowledge, a new behavior of PIP2 proposed to occur in cells, in which local multivalent metal ions, lipid compositions, and various binding proteins could greatly influence PIP2 properties. In turn, these different pools of PIP2 could further regulate cellular events.

Domains of the Hepatitis B Virus Small Surface Protein S Mediating Oligomerization.

During hepatitis B virus (HBV) infections, subviral particles (SVP) consisting only of viral envelope proteins and lipids are secreted. Heterologous expression of the small envelope protein S in mammalian cells is sufficient for SVP generation. S is synthesized as a transmembrane protein with N-terminal (TM1), central (TM2), and hydrophobic C-terminal (HCR) transmembrane domains. The loops between TM1 and TM2 (the cytosolic loop [CL]) and between TM2 and the HCR (the luminal loop [LL]) are located in the cytosol and the endoplasmic reticulum (ER) lumen, respectively. To define the domains of S mediating oligomerization during SVP morphogenesis, S mutants were characterized by expression in transiently transfected cells. Mutation of 12 out of 15 amino acids of TM1 to alanines, as well as the deletion of HCR, still allowed SVP formation, demonstrating that these two domains are not essential for contacts between S proteins. Furthermore, the oligomerization of S was measured with a fluorescence-activated cell sorter (FACS)-based Förster resonance energy transfer (FRET) assay. This approach demonstrated that the CL, TM2, and the LL independently contributed to S oligomerization, while TM1 and the HCR played minor roles. Apparently, intermolecular homo-oligomerization of the CL, TM2, and the LL drives S protein aggregation. Detailed analyses revealed that the point mutation C65S in the CL, the mutation of 13 out of 19 amino acids of TM2 to alanine residues, and the simultaneous replacement of all 8 cysteine residues in the LL by serine residues blocked the abilities of these domains to support S protein interactions. Altogether, specific domains and residues in the HBV S protein that are required for oligomerization and SVP generation were defined.IMPORTANCE The small hepatitis B virus envelope protein S has the intrinsic ability to direct the morphogenesis of spherical 20-nm subviral lipoprotein particles. Such particles expressed in yeast or mammalian cells represent the antigenic component of current hepatitis B vaccines. Our knowledge about the steps leading from the initial, monomeric, transmembrane translation product of S to SVP is very limited, as is our information on the structure of the complex main epitope of SVP that induces the formation of protective antibodies after vaccination. This study contributes to our understanding of the oligomerization process of S chains during SVP formation and shows that the cytoplasmic loop, one membrane-embedded domain, and the luminal loop of S independently drive S-S oligomerization.

De novo design of selective Sortase-A inhibitors: Synthesis, structural and in vitro characterization

Abstract Novel molecules were designed against Staphylococcus aureus sortase A, an imperative and vital target involved in the bacterial virulence. Structure guided de novo designing was performed using different chemical fragments. These fragments were extended based on the requirements of the binding cavity. All the compounds in its binding orientation has formed good interaction network with amino acids HIS 120 and ARG 197. Molecules designed contain indole-chroman and indolin-chroman moieties and their docking scores were ranging from −6.92 to −5.67. Designed molecules were selected for synthesis and tested in vitro against Staphylococcus aureus sortase A using Fluorescence resonance energy transfer assay. The highest active molecule KK4 ((Z)-3-((5-nitro-1H-indol-3-yl) methylene) chroman-2,4‑dione) has IC50 of 3.4 µM. This molecule with indole chroman moiety is suitable candidate that can be used as a prospective lead against Staphylococcus aureus sortase A.

Polymerase-assisted fluorescence resonance energy transfer (FRET) assay for simultaneous detection of binary viral sequences

The analysis of a specific sequence of nucleic acids enables identification of pathogens and the diagnosis of human genetic disorders. This emphasises the need to develop methods of detecting nucleic acids, particularly in a multiplex format, that yield a decisive conclusion for clinical interpretation. Herein, we introduce a polymerase-assisted fluorescence resonance energy transfer (FRET) assay to simultaneously analyse binary viral genes that are characteristic of hemagglutinin and neuraminidase in influenza A virus. The approach takes advantage of the formation of an X-shaped four-way DNA junction (4WJ), thus enabling a selective response to the binary targets of specific sequences. Polymerase drives the cycling of the target complex and incorporates 2′-deoxyuridine-5-triphosphate (dUTP) labelled with Texas Red (TR-dUTP) into the double-stranded DNA (dsDNA) that is produced, which induces FRET to produce a sensing output. Crucially, the mechanism relies on a DNA hairpin instead of a molecular beacon, which substantially increases the simplicity of the assay and reduces its cost. The results revealed that the lowest detectable concentration was approximately 2 pM. The donor−acceptor distance was approximately 7 Å and independent of the ratio of TR-dUTP to 2′-deoxythymidine-5-triphosphate (dTTP). An ‘off–on’ assay operating in AND gate mode was demonstrated and is potentially useful for the fast diagnosis and subtyping of influenza viruses.

Heteromerization of \u03bc-opioid receptor and cholecystokinin B receptor through the third transmembrane domain of the \u03bc-opioid receptor contributes to the anti-opioid effects of cholecystokinin octapeptide

Activation of the cholecystokinin type B receptor (CCKBR) by cholecystokinin octapeptide (CCK-8) inhibits opioid analgesia. Chronic opiate treatment leads to an increase in the CCK-8 concentration and thus enhances the antagonism of CCK-8 against opioid analgesia; the underlying molecular mechanisms remain of great interest. In the present study, we validated the colocalization of the μ-opioid receptor (MOR) and CCKBR in pain signal transmission-related spinal cord dorsal horn and dorsal root ganglion neurons of rats. Co-immunoprecipitation (Co-IP) and fluorescence lifetime-imaging-microscopy-based fluorescence resonance energy transfer (FLIM-FRET) assays showed that MOR heteromerized with CCKBR directly in transfected HEK293 cells. Combined with MOR mutant construction, the third transmembrane domain of MOR (TM3MOR) was demonstrated to participate in heteromerization with CCKBR. Receptor ligand binding, ERK phosphorylation and cAMP assays showed that MOR heteromerization with CCKBR weakened the activity of MOR. A cell-penetrating interfering peptide consisting of TM3MOR and TAT (a transactivator of HIV-1) sequences from the N terminal to the C terminal disrupted the MOR–CCKBR interaction and restored the activity of MOR in transfected HEK293 cells. Furthermore, intrathecal application of the TM3MOR-TAT peptide alleviated CCK-8-injection-induced antagonism to morphine analgesia in rats. These results suggest a new molecular mechanism for CCK-8 antagonism to opioid analgesia in terms of G-protein-coupled receptor (GPCR) interaction through direct heteromerization. Our study may provide a potential strategy for pain management with opioid analgesics.

Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells

G protein–coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase–mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype–biased ligands.