Fragment Complementation Assay Research Articles

Abstract 3045: PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer

Abstract Peroxisome proliferator activated receptor alpha (PPARα) is a transcription factor and master regulator of fatty acid oxidation. PPARα ligands modulate the switch between co-activator and co-repressor transcription complexes at PPARα-controlled target genes. In addition, PPARα has been shown to transrepress NF-κB signaling and inhibit angiogenesis. Renal cell carcinoma (RCC) expresses high levels of PPAR-α and is a highly angiogenic cancer. Current frontline treatments for RCC include chemotherapy, anti-angiogenics, and immunotherapy but are limited by the immune suppressive state of the tumor microenvironment. TPST-1120 is a novel PPARα antagonist for the treatment of cancer patients that binds in the ligand binding domain, positioning the AF-2 activation helix in an inactive conformation. The inactive conformation has a lower affinity for co-activator motifs and reduces activation of PPARα regulated genes. In a CHO-based reporter cell line, TPST-1120 potently competed against the PPARα-specific agonist GW7647 (IC50 = 36 nM at 20 nM GW7647) and an endogenous PPARα agonist oleoylethanolamide (OEA; IC50 = 15 nM at 30 µM OEA). Furthermore, in an enzyme fragment complementation assay, we demonstrated that TPST-1120 inhibited agonist-induced binding of PPARα to MED1 by destabilizing the co-activator complex thereby stabilizing the inactive conformation of PPAR-α. In a murine model of renal cell adenocarcinoma (RENCA), TPST-1120 treatment for 12 days (30 mg/kg qd) reduced tumor growth by 52% as a monotherapy (p<0.0001). Consistent with these findings, TPST-1120 inhibited tumor angiogenesis and proliferation as quantified by Ki67 levels. Combination treatment with current frontline therapeutics resulted in synergistic tumor inhibition of 74% with anti-PD1 (200 ug Q3D for 12 days) and 81% with cabozantinib (15 mg/kg QD for 15 days) (p<.0001). Flow cytometry-based analysis of tumors demonstrated increased CD8+ T cell infiltration in the TME as well as decreases in M2 and elevations in M1 macrophages. Importantly, we demonstrate that TPST-1120 can reverse an immunosuppressive class of immune cells to promote anti-tumor immunity and anti-angiogenesis in kidney cancer in the absence of overt toxicity. These data support advancement of TPST-1120 into RCC. Citation Format: Michael Gillespie, Valerie Chen, Isabella Howard, Keira Smith, Dave Freund, Nathan Standifer, Dara Burdette, Thomas W. Dubensky, Sam Whiting, Dipak Panigrahy. PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3045.

Creation of Artificial Luciferase 60s from Sequential Insights and Their Applications to Bioassays.

In this study, a series of new artificial luciferases (ALucs) was created using sequential insights on missing peptide blocks, which were revealed using the alignment of existing ALuc sequences. Through compensating for the missing peptide blocks in the alignment, 10 sibling sequences were artificially fabricated and named from ALuc55 to ALuc68. The phylogenetic analysis showed that the new ALucs formed an independent branch that was genetically isolated from other natural marine luciferases. The new ALucs successfully survived and luminesced with native coelenterazine (nCTZ) and its analogs in living mammalian cells. The results showed that the bioluminescence (BL) intensities of the ALucs were interestingly proportional to the length of the appended peptide blocks. The computational modeling revealed that the appended peptide blocks created a flexible region near the active site, potentially modulating the enzymatic activities. The new ALucs generated various colors with maximally approximately 90 nm redshifted BL spectra in orange upon reaction with the authors' previously reported 1- and 2-series coelenterazine analogs. The utilities of the new ALucs in bioassays were demonstrated through the construction of single-chain molecular strain probes and protein fragment complementation assay (PCA) probes. The success of this study can guide new insights into how we can engineer and functionalize marine luciferases to expand the toolbox of optical readouts for bioassays and molecular imaging.

Metabolic modulation of CtBP dimeric status impacts the repression of DNA damage repair genes and the platinum sensitivity of ovarian cancer.

Platinum drug-based chemotherapy plays a dominant role in OC (ovarian cancer) treatment. The expression of DNA damage repair (DDR) genes is critical in distinguishing drug-sensitive and drug-refractory patients, as well as in the development of drug resistance in long-term treated patients. CtBP is a highly expressed oncogene in OC and was found to repress DDR genes expression in our previous study. In the present study, the formation of CtBP dimers in live cells was studied, and the functional differences between monomeric and oligomeric CtBP were explored by CHIP-seq and RNA-seq. Besides, the dynamics of CtBP dimer formation in response to the metabolic modulation were investigated by the protein fragment complementation (PCA) assays. We show that dimerized CtBP, but not the dimerization-defective mutant, binds to and represses DDR gene expression in OC cells. Treatment of the mice tumors grown from engrafted OC cells by cisplatin disclosed that high-level CtBP expression promotes the CtBP dimerization and increases the therapeutic effect of cisplatin. Moreover, the CtBP dimerization is responsive to the intracellular metabolic status as represented by the free NADH abundance. Metformin was found to increase the dimerization of CtBP and potentiate the therapeutic effect of cisplatin in a CtBP dimerization-dependent manner. Our data suggest that the CtBP dimerization status is a potential biomarker to predict platinum drug sensitivity in patients with ovarian cancer and a target of metformin to improve the therapeutic effect of platinum drugs in OC treatment.

Abstract 3932: Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is)

Abstract CC-99282 is a novel CELMoD® agent that was optimized for activity in non-Hodgkin lymphoma (NHL). CC-99282 potently degrades Ikaros and Aiolos, resulting in enhanced antiproliferative, apoptotic, and immune-stimulatory activity in diffuse large B-cell lymphoma (DLBCL) models, including those with acquired chemoresistance (Lopez-Girona A, et al. Hematol Oncol. 2021). In lymphocytes, Ikaros negatively regulates gene expression via histone modifications, including polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) (Oravecz A, et al. Nat Commun. 2015); in NHL, these epigenetic mechanisms are unclear. Using DLBCL models, we explored the relationship between CC-99282 activity and epigenetic status, and the mechanism of synergy of CC-99282 treatment and inhibition of EZH2, a PRC2 component. Baseline characteristics of human DLBCL lines and effects of CC-99282 ± tazemetostat (TAZ), a representative EZH2i, were evaluated in vitro and in vivo by chromatin immunoprecipitation sequencing, gene expression, flow cytometry, immunoblotting, enzyme fragment complementation assays, and/or CRISPR/Cas9 gene editing. Analysis of Ikaros/Aiolos degradation in > 20 DLBCL cell lines showed their loss is necessary, but not sufficient for CC-99282 efficacy. Evaluation of baseline histone marks showed that the sensitive cell lines exhibit aberrant, higher H3K27me3 coverage at promoter regions of expressed genes. This suggests a direct correlation between H3K27me3 status at these regions and CC-99282 sensitivity. In T cells, Oravecz et al. found that loss of Ikaros reduces H3K27me3 at specific chromatin sites due to its interaction with PRC2. We confirmed that these affected regions are enriched in genes upregulated upon CC-99282 treatment in DLBCL cell lines, suggesting a similar role for Ikaros in DLBCL. Pathway analysis following CC-99282 or TAZ treatment demonstrated high overlap between pathways altered by both agents. CC-99282 + TAZ combined demonstrated additive and/or synergistic antiproliferative and apoptotic effects in DLBCL cell lines. This combination did not alter Ikaros degradation or overall H3K27me3 status but increased downstream effects. Results were confirmed by CRISPR/Cas9 knockout competition assays with EZH2 and other PRC2 components. This effect was independent of cell of origin, EZH2 mutational status, or degree of CC-99282 sensitivity. Synergy was confirmed using the SU-DHL6 and DB xenograft models that are intrinsically resistant to EZH2is and CC-99282, respectively. Combination treatment yielded durable responses and tumor-free animals. Collectively, these data suggest that Ikaros could act as an epigenetic modulator through PRC2 recruitment and support the combination of CC-99282 with EZH2is in NHL (NCT03930953) to favor broad and durable clinical responses. Citation Format: Soraya Carrancio, Celia Fontanillo, Ryan Galasso, Martino Colombo, Scott Wood, Carla Guarinos, Alejandro Panjkovich, Diana Jankeel, Adam Blattler, Preethi Janardhanan, Matthew Groza, Jim Leisten, Rama Krishna Narla, Antonia Lopez-Girona, Daniel W. Pierce. Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3932.

CRISPR-Mediated Enzyme Fragment Complementation Assay for Quantification of the Stability of Splice Isoforms.

Small-molecule splicing modulators exemplified by an FDA-approved drug, risdiplam, are a new pharmacological modality for regulating the expression and stability of splice isoforms. We report a CRISPR-mediated enzyme fragment complementation (EFC) assay to quantify the splice isoform stability. The EFC assay harnessed a 42 amino acid split of a β-galactosidase (designate α-tag), which could be fused at the termini of the target genes using CRISPR/cas9. The α-tagged splice isoform would be quantified by measuring the enzymatic activity upon complementation with the rest of β-galactosidase. This EFC assay retained all the sequences of introns and exons of the target gene in the native genomic environment that recapitulates the cell biology of the diseases of interest. For a proof-of-concept, we developed a CRISPR-mediated EFC assay targeting the exon 7 of the survival of motor neuron 2 (SMN2) gene. The EFC assay is compatible with 384-well plates and robustly quantified the splicing modulation activity of small molecules. In this study, we also discovered that a coumarin derivative, compound 4, potently modulated SMN2 exon 7 splicing at as low as 1.1 nM.

Characterization of the MurT/GatD complex in Mycobacterium tuberculosis towards validating a novel anti-tubercular drug target.

ObjectivesIdentification and validation of novel therapeutic targets is imperative to tackle the rise of drug resistance in tuberculosis. An essential Mur ligase-like gene (Rv3712), expected to be involved in cell-wall peptidoglycan (PG) biogenesis and conserved across mycobacteria, including the genetically depleted Mycobacterium leprae, was the primary focus of this study.MethodsBiochemical analysis of Rv3712 was performed using inorganic phosphate release assays. The operon structure was identified using reverse-transcriptase PCR and a transcription/translation fusion vector. In vivo mycobacterial protein fragment complementation assays helped generate the interactome.ResultsRv3712 was found to be an ATPase. Characterization of its operon revealed a mycobacteria-specific promoter driving the co-transcription of Rv3712 and Rv3713. The two gene products were found to interact with each other in vivo. Sequence-based functional assignments reveal that Rv3712 and Rv3713 are likely to be the mycobacterial PG precursor-modifying enzymes MurT and GatD, respectively. An in vivo network involving Mtb-MurT, regulatory proteins and cell division proteins was also identified.ConclusionsUnderstanding the role of the enzyme complex in the context of PG metabolism and cell division, and the implications for antimicrobial resistance and host immune responses will facilitate the design of therapeutics that are targeted specifically to M. tuberculosis.

Pharmacological Activation of Nrf2 by Rosolic Acid Attenuates Endoplasmic Reticulum Stress in Endothelial Cells.

Endoplasmic reticulum (ER) plays a key role in the folding, modification, and trafficking of proteins. When the homeostasis of the ER is disturbed, un/misfolded proteins accumulate in the ER which leads to ER stress. Sustained ER stress results in apoptosis, which is associated with various diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor in redox homeostasis by regulating various genes associated with detoxification and cell-protective mechanisms. We found that Rosolic acid (RA) treatment dose-dependently activates Nrf2 in endothelial cells using the enzyme fragment complementation assay. The cytoprotective role of RA against ER stress-induced endothelial apoptosis and its molecular mechanism was explored in the present study. The Nrf2 and its target genes, as well as ER stress marker expressions, were measured by qPCR in ER stress-exposed endothelial cells. The contribution of Nrf2 in RA-mediated defense mechanism in endothelial cells was established by knockout studies using Nrf2-CRISPR/Cas9. The treatment with RA to ER stress-induced endothelial cells exhibited activation of Nrf2, as demonstrated by Nrf2 translocation and reduction of ER stress markers. We found that the Nrf2 knockout sensitized the endothelial cells against ER stress, and further, RA failed to mediate its cytoprotective effect. Proteomic studies using LC-MS/MS revealed that among the 1370 proteins detected, we found 296 differentially regulated proteins in ER stress-induced endothelial cells, and RA administration ameliorated 71 proteins towards the control levels. Of note, the ER stress in endothelial cells was attenuated by the treatment with the RA, suggesting the role of the Nrf2 activator in the pathological conditions of ER stress-associated diseases.

A Multipronged Screening Approach Targeting Inhibition of ETV6 PNT Domain Polymerization

ETV6 is an ETS family transcriptional repressor for which head-to-tail polymerization of its PNT domain facilitates cooperative binding to DNA by its ETS domain. Chromosomal translocations frequently fuse the ETV6 PNT domain to one of several protein tyrosine kinases. The resulting chimeric oncoproteins undergo ligand-independent self-association, autophosphorylation, and aberrant stimulation of downstream signaling pathways, leading to a variety of cancers. Currently, no small-molecule inhibitors of ETV6 PNT domain polymerization are known and no assays targeting PNT domain polymerization have been described. In this study, we developed complementary experimental and computational approaches for identifying such inhibitory compounds. One mammalian cellular approach utilized a mutant PNT domain heterodimer system covalently attached to split Gaussia luciferase fragments. In this protein–fragment complementation assay, inhibition of PNT domain heterodimerization reduces luminescence. A yeast assay took advantage of activation of the reporter HIS3 gene upon heterodimerization of mutant PNT domains fused to DNA-binding and transactivation domains. In this two-hybrid screen, inhibition of PNT domain heterodimerization prevents cell growth in medium lacking histidine. The Bristol University Docking Engine (BUDE) was used to identify virtual ligands from the ZINC8 library predicted to bind the PNT domain polymerization interfaces. More than 75 hits from these three assays were tested by nuclear magnetic resonance spectroscopy for binding to the purified ETV6 PNT domain. Although none were found to bind, the lessons learned from this study may facilitate future approaches for developing therapeutics that act against ETV6 oncoproteins by disrupting PNT domain polymerization.

A Split Methyl Halide Transferase AND Gate That Reports by Synthesizing an Indicator Gas.

Monitoring microbial reactions in highly opaque or autofluorescent environments like soils, seawater, and wastewater remains challenging. To develop a simple approach for observing post-translational reactions within microbes situated in environmental matrices, we designed a methyl halide transferase (MHT) fragment complementation assay that reports by synthesizing an indicator gas. We show that backbone fission within regions of high sequence variability in the Rossmann domain yields split MHT (sMHT) AND gates whose fragments cooperatively associate to synthesize CH3Br. Additionally, we identify a sMHT whose fragments require fusion to pairs of interacting partner proteins for maximal activity. We also show that sMHT fragments fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR display significantly enhanced CH3Br production in the presence of rapamycin. This gas production is reversed in the presence of the competitive inhibitor of FKBP12/FKPB dimerization, indicating that sMHT is a reversible reporter of post-translational reactions. This sMHT represents the first genetic AND gate that reports on protein-protein interactions via an indicator gas. Because indicator gases can be measured in the headspaces of complex environmental samples, this assay should be useful for monitoring the dynamics of diverse molecular interactions within microbes situated in hard-to-image marine and terrestrial matrices.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

SummaryLanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Correction: Sensitive detection of protein ubiquitylation using a protein fragment complementation assay.

There was an error in J. Cell Sci. (2020) 133, jcs240093 (doi:10.1242/jcs.240093).In their article, the authors omitted to mention ubiquitin-mediated fluorescence complementation (UbFC), a method previously designed to probe the conjugation of ubiquitin or ubiquitin-like proteins (Ubls) to their substrates (Fang and Kerppola, 2004; Sung et al, 2013). UbFC and NUbiCA are similar in that both methods rely on a protein-fragment complementation assay to detect Ubl conjugation, but the reporters (fluorescent proteins for UbFC, the NanoLuc luciferase for NUbiCA) and assay conditions (live or fixed cells for UbFC, purified proteins for NUbiCA) are different. UbFC is appealing since it enables direct visualization of the subcellular localization of Ubl conjugates in living cells, which is not possible with NUbiCA. Yet, the irreversible assembly and slow maturation of fluorescent proteins (Hu et al., 2002; Kodama and Hu, 2012) precludes real-time monitoring of Ubl conjugation and deconjugation events, which may complicate the interpretation of UbFC results, especially for short-lived or dynamically modified conjugates. Other approaches based on FRET and BRET have also been devised to probe ubiquitylation of proteins in living cells, but their sensitivity has not yet been carefully assessed (Ganesan et al., 2006; Batters et al., 2010; Riching et al., 2018).The authors apologise to readers for this omission, which does not impact the results or conclusions of the paper.

Sensitive detection of protein ubiquitylation using a protein fragment complementation assay.

Ubiquitylation is a reversible post-translational protein modification that regulates a multitude of cellular processes. Detection of ubiquitylated proteins is often challenging because of their low abundance. Here, we present NUbiCA, a sensitive protein-fragment complementation assay to facilitate the monitoring of ubiquitylation events in cultured cells and model organisms. Using yeast as a model system, we demonstrate that NUbiCA enables accurate monitoring of mono- and polyubiquitylation of proteins expressed at endogenous levels. We also show that it can be applied to decipher the topology of ubiquitin conjugates. Moreover, we assembled a genome-wide collection of yeast strains ready to investigate the ubiquitylation of proteins with this new assay. This resource will facilitate the analysis of local or transient ubiquitylation events that are difficult to detect with current methods.

Correction to: Chaperone-substrate interactions monitored via a robust TEM-1 β-lactamase fragment complementation assay.

In the original publication of the article, under the "Acknowledgement" section, the Grant No. 31611011097 should read as No. 31661143021.

Gymnema montanum improves endothelial function via inhibition of endoplasmic reticulum stress by activating Nrf2 signaling

Objective: To investigate the effects of Gymnema montanum leaf extract against endoplasmic reticulum (ER) stress-induced toxicity in endothelial cells. Methods: The immortalized endothelial hybrid cell, EA.hy926 was treated with different concentrations of Gymnema montanum leaf extract (0-100 μg/mL) and the ER stress inducer, tunicamycin. The cytotoxicity was assessed by MTT as well as lactate dehydrogenase and malondialdehyde levels were determined. The levels of ER stress markers, GRP78 and CHOP were analysed by Western blot assay. The Gymnema montanum leaf extract-mediated activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was assessed by cell-based luciferase enzyme fragment complementation assay and antioxidant responsive element driven luciferase reporter assay. The levels of phosphoproteins of the MAPK pathway were analyzed using the Bioplex system. Results: A dose-dependent cytoprotective effect of Gymnema montanum leaf extract was observed in tunicamycin-induced toxicity. Gymnema montanum leaf extract significantly reduced lactate dehydrogenase activity and malondialdehyde levels in ER stress-induced endothelial cells. It also suppressed ER stress markers dose dependently and inhibited the phosphorylation of JNK, ERK, MEK and p38 MAPK in tunicamycin-induced endothelial cells. Moreover, Gymnema montanum leaf extract increased the expression of Nrf2 and its downstream targets in endothelial cells. Conclusions: Gymnema montanum leaf extract attenuates ER stress by increasing the expression of Nrf2 and its downstream genes.

PS1441 A NOVEL ACTIVATING TPOR MUTATION IN ESSENTIAL THROMBOCYTOPENIA POINTS TO AN ACTIVATING EFFECT OF TRYPTOPHAN AND AROMATICS AT THE OUTSET OF THE TRANSMEMBRANE DOMAIN

Background:Thrombopoietin receptor (TpoR) is a type‐I dimeric cytokine receptor that regulates platelet formation and hematopoietic stem cell renewal. Tpo binding mediates TpoR dimerization, which imparts a specific orientation on the receptor transmembrane (TM) and intracellular (IC) domains that leads to JAK2 trans‐phosphorylation. Mutations in key amino acids in the TpoR TM or cytosolic juxtamembrane (JM) domains lead to myeloproliferative neoplasms. The study of such W515K/L/A/R/S mutants at the cytosolic TM‐JM border previously demonstrated the critical role of tryptophan (Trp) buried in the phospholipid headgroup region in orienting the receptor and preventing the favorable interfacial contacts that lead to JAK2 activation.Aims:In a patient with essential thrombocytopenia (ET) we identified by targeted sequencing as the only alteration a heterozygous TpoR L498W‐H499C mutation. These residues are located at the extracellular JM‐TM border and this was provocative as H499 is the binding site for the small molecule TpoR agonist eltrombopag. Moreover, the structure of this region is important for regulating TpoR function. Our aim was to assess the effects of this double mutation in TpoR and, if active, to elucidate the mechanism of activation. We extended this study to other cytokine receptors.Methods:We first tested the ability of the single L498W and H499C mutants and the double mutants found in the patient to induce basal and ligand‐dependent activation of STAT5 in a dual luciferase reporter assay. We next performed Trp‐scanning mutagenesis around L498. In addition, we addressed whether an aromatic residue was required at L498 for autonomous activation and performed targeted mutagenesis. We then assessed TpoR dimerization by cysteine‐based cross‐linking and NanoBiT protein fragment complementation assays and constructed a TM‐intracellular model of TpoR (i.e. without the extracellular domain), to test the requirement of the extracellular domain in the activation process. Simultaneously, the homologous mutations were also introduced in the murine TpoR (L491W‐L492C). Finally, we performed a WES analysis of granulocytic DNA of the index patient to exclude other driver mutations.Results:The TpoR L498W‐H499C mutation identified in an ET patient is constitutively active due to the L498W single mutation. Only tyrosine and phenylalanine among tested residues induced constitutive TpoR activation, although weaker, indicating that the aromaticity and/or p interactions lead to self‐activation. This is specific for position 498 as tryptophan scanning of the region did not give another active mutant. Dimerization assays indicated ligand‐independent dimerization of the TM domain for both L498W and H499C. Finally, we showed that the L491W‐L492C mutation in murine TpoR also leads to constitutive activation. Of interest, W498 (activating) and W515 (preventing activation) belong to the same helical face of the TM‐JM region, capping the TM domain. These results suggest that position 498 allosterically overrides the inhibitory effects of W515, suggesting the region around 498 is an allosteric site for TpoR function.Summary/Conclusion:We show that aromatic mutations at position 498 at the extracellular JM region of the human TpoR are responsible for constitutive activation. This leads to dimerization in a productive conformation for JAK2 indicating that this particular region of TpoR and possibly of other receptors could be targets for small molecules. This effect is independent of, but proximal to the H499 residue, which is bound by eltrombopag.

Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

BackgroundThe most commonly occurring cancer mutations, including oncogenes such as MYC, Ras and PIK3C, are found in signal transductions pathways feeding into the translational machinery. A broad range of translation initiation factors are also commonly found to be either amplified or mis-regulated in tumours, including eIF4E (elongation initiation factor 4E). eIF4E is a subunit of the eIF4F protein initiation complex and required for its recruitment. Here we measure the formation of the eIF4F complex through interactions of eIF4E and eIF4G subunits, and the effect of oncogenic signalling pathways on complex formation.ResultsWe developed a protein fragment complementation (PCA) assay that can accurately measure the status of the eIF4E-eIF4G interaction in cells and quantify the signalling flux through the RAS/ERK and PI3K/AKT pathways regulating eIF4F assembly. Complex disruption induced by inhibition of either pathway was shown to be a function of the phosphorylation status of 4EBP1, a key mediator of eIF4F assembly that interacts directly with eIF4E, confirming 4EBP1’s ability to integrate multiple signals affecting cap-dependent translation. Maximal measured disruption of the eIF4F complex occurred under combined mTORC1 and mTORC2 inhibition, whilst combined inhibition of both RAS/ERK and PI3K/AKT pathways in parallel resulted in greater inhibition of eIF4F formation than individually. v-Myc-mediated resistance to dual mTORC/PI3K inhibition was also principally demonstrated to depend on the lack of competent 4EBP1 available in the cell to bind eIF4E.ConclusionsWe show that 4EBP1 is a critical regulator of the mitogen responsive RAS/ERK and PI3K/AKT pathways and a key transducer of resistance mechanisms that affect small molecule inhibition of these pathways, principally by attenuating their effects on cap-dependent translation. These findings highlight the importance of highly efficacious direct inhibitors of eIF4E and eIF4F assembly, which could potentially target a wide spectrum of tumours containing differing mutations that effect these pathways and which confer chemo-resistance.

In vivo assembly and trafficking of olfactory Ionotropic Receptors

BackgroundIonotropic receptors (IRs) are a large, divergent subfamily of ionotropic glutamate receptors (iGluRs) that are expressed in diverse peripheral sensory neurons and function in olfaction, taste, hygrosensation and thermosensation. Analogous to the cell biological properties of their synaptic iGluR ancestors, IRs are thought to form heteromeric complexes that localise to the ciliated dendrites of sensory neurons. IR complexes are composed of selectively expressed ‘tuning’ receptors and one of two broadly expressed co-receptors (IR8a or IR25a). While the extracellular ligand-binding domain (LBD) of tuning IRs is likely to define the stimulus specificity of the complex, the role of this domain in co-receptors is unclear.ResultsWe identify a sequence in the co-receptor LBD, the ‘co-receptor extra loop’ (CREL), which is conserved across IR8a and IR25a orthologues but not present in either tuning IRs or iGluRs. The CREL contains a single predicted N-glycosylation site, which we show bears a sugar modification in recombinantly expressed IR8a. Using the Drosophila olfactory system as an in vivo model, we find that a transgenically encoded IR8a mutant in which the CREL cannot be N-glycosylated is impaired in localisation to cilia in some, though not all, populations of sensory neurons expressing different tuning IRs. This defect can be complemented by the presence of endogenous wild-type IR8a, indicating that IR complexes contain at least two IR8a subunits and that this post-translational modification is dispensable for protein folding or complex assembly. Analysis of the subcellular distribution of the mutant protein suggests that its absence from sensory cilia is due to a failure in exit from the endoplasmic reticulum. Protein modelling and in vivo analysis of tuning IR and co-receptor subunit interactions by a fluorescent protein fragment complementation assay reveal that the CREL N-glycosylation site is likely to be located on the external face of a heterotetrameric IR complex.ConclusionsOur data reveal an important role for the IR co-receptor LBD in control of intracellular transport, provide novel insights into the stoichiometry and assembly of IR complexes and uncover an unexpected heterogeneity in the trafficking regulation of this sensory receptor family.

Using CRISPR/Cas9 and NanoLuc to investigate “endogenous” CXCR4 ligand binding, internalization and β‐arrestin2 recruitment

Ligand binding, receptor internalisation and receptor‐protein interactions are all vital components to receptor signalling that can be investigated using bioluminescence resonance energy transfer (BRET) or fragment complementation assays. However a fundamental limitation of these techniques is the requirement for exogenous expression of fusion proteins, which precludes the application of these methods to study endogenous proteins. Recently, we have demonstrated that BRET can be used to measure protein‐protein interactions when the luciferase NanoLuc was inserted into the genome by CRISPR/Cas9 engineering. Using NanoBRET and the NanoBiT luciferase complementation here we aimed to investigate ligand binding, receptor internalisation and receptor‐β‐arrestin2 interactions when the CXCR4 and/or interacting partners were expressed under endogenous promotion.CRISPR/Cas9‐mediated homology‐directed repair was used to insert Nluc or NanoBiT fragments into the CXCR4, ARRB2 or CXCL12 genomic loci in HEK293 cells. For NanoBRET ligand binding studies, cells expressing HiBiT/ or Nluc/CXCR4 were incubated for 1hr with CXCL12‐AF647 in the absence or presence of AMD3100 (10 μM). Alternatively cells expressing gene‐edited CXCL12/HiBiT were transfected with SNAP/CXCR4, labelled with cell surface SNAP‐AF488 and incubated with or without AMD3100 (10 pM‐10 μM). For internalisation assays cells were incubated with unlabelled ligands only. For β‐arrestin recruitment assays cells expressing both gene‐edited CXCR4/LgBiT and β‐arr2/SmBiT, or cells expressing one gene‐edited protein transfected with the other, were stimulated with CXCL12 (1 pM – 1 μM) and luminescence measured for 1 hr. Luminescence was generated by addition of furimazine and for cells expressing HiBiT 10 nM LgBiT and was measured on a PHERAstar plate reader (BMG).Binding affinities (Kd) of CXCL12‐AF647 were obtained via NanoBRET saturation binding experiments at Nluc/CXCR4 or HiBiT/CXCR4 expressed under endogenous promotion (nM; 22.74 ± 2.05 and 23.47 ± 4.16, n=4 respectively). Binding of gene edited CXCL12/HiBiT to SNAP/CXCR4 was displaced by AMD3100 (pIC50, 7.09 ± 0.16, n=5). In internalization experiments application of CXCL12 to cells expressing gene‐edited HiBiT/CXCR4 resulted in a concentration dependent decrease in luminescence (pEC50, 8.67 ± 0.27, n=5) suggestive of internalization. In cells expressing gene‐edited CXCR4/LgBiT, β‐arr2/SmBiT or both NanoBiT fragments application of CXCL12 resulted in a concentration dependent increase in luminescence respectively that was inhibited by AMD3100, indicative of ligand induced β‐arrestin2 recruitment to CXCR4Using CRISPR/Cas9 mediated genome engineering, NanoBRET and NanoBiT complementation here we have shown that multiple facets of GPCR activation, desensitisation and signalling can be investigated using proteins expressed under endogenous promotion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

JAK2 V617F Dimerizes Homodimeric Cytokine Receptors Cytosolic Domains By Requiring Pseudokinase Domain Residues That Promote JAK2 Dimerization and Oncogenic Activity

JAK2 plays roles in several signaling pathways by binding to multiple distinct cytokine receptors. Dysfunction of JAK2 signaling is associated with hematopoietic malignancies. The acquired mutation JAK2 V617F, which constitutively activates JAK2 kinase, is the most common molecular event associated with myeloproliferative disorders.Using a combination of bioluminescence energy transfer and protein fragment complementation assays we show that V617F located in the pseudokinase domain of JAK2 plays an important role on JAK2 dimerization via the C-terminal kinase domains. Using NanoBRET we show that JAK2 dimerization via the C-terminal domains is reduced by single mutations (E596R 1, F595A 2, A598F and F537A) that in the context of JAK2 V617F revert the mutant to wild type phenotype with respect to signaling by homodimeric erythropoietin and thrombopoietin receptors (EpoR and TpoR). Thus, residues that are required for V617F activation strengthen JAK2 dimerization via C-terminal kinase domains, implicating the pseudokinase domain in global JAK2 dimerization and suggesting that part of the V617F activation mechanism relies on dimerization of mutated pseudokinase domains.Using NanoBiT protein fragment complementation assay, we explored the effects of JAK2 V617F on dimerization of the cytosolic ends of cytokine receptors. We show that JAK2 V617F promotes strong enhancement of dimerization of EpoR and TpoR cytosolic domains, when compared to wild type JAK2. This result is unexpected for EpoR that was shown to exist in a preformed dimer configuration at a significantly higher degree than TpoR3-4. Nevertheless our results indicate that JAK2 V617F enhances close apposition of cytosolic domains of EpoR and TpoR, which could occur within preformed complexes or by a switch from monomer to dimer configuration induced by JAK2 V671F. This enhancement of dimerization is prevented when JAK2 V617F carries any of the reverting mutations (E596R, F595A, A598F, F537A) that prevent V617F activation. We also detected enhanced EpoR dimerization for exon 12 JAK2 mutation and JAK2 T875N, but not for other JAK2 mutants that are involved in lymphoid malignancies. The signals we detect are not simply the result of signaling and clustering in vesicles or internalization, as inhibition of signaling via Ruxolitinib does not prevent the dimerization effects on receptors. Our results indicate that rather than preformed dimeric receptors aiding in JAK2 V617F activation5, JAK2 V617F promotes close apposition of cytosolic domains of dimeric receptors. This result is in agreement with a recent study that showed that JAK2 V617F requires a closer proximity of EpoR chains than signaling by wild type JAK26. Such differences in dimeric requirements might open new therapeutic avenues for JAK2 V617F inhibition.We extended these explorations to other cytokine receptors utilizing JAK2 or JAK1 belonging to type I and type II subfamilies. Results on JAK2 are transposable to JAK1 for homologous mutations. Most receptors tested, such as EpoR, TpoR, G-CSFR, IL2, IL9 and type I interferon respond to their cytokine when JAK2 (or JAK1 for those utilizing JAK1) carries mutations known to inhibit JAK2 V617F (or JAK1 V658F, homologous to JAK2 V617F). One prominent exception is interferon γ-receptor (IFNGR), which is significantly impaired in its response to IFNγ when JAK2 and JAK1 carry such mutations. We show that homodimeric interactions within the heterotetrameric IFNGR recruiting JAK2 and JAK1 explain this inhibitory effect.Thus, it appears that a specific region around pseudokinase domain helix αC, which is required for JAK2 V617F and JAK1 V658F oncogenic activation, is also critical for relaying normal IFNγ signaling via its tetrameric receptor complex, which appears to require enhanced dimerization between JAK1 and JAK2 pseudokinase domains. Our results provide critical information for the design of strategies aiming to inhibit JAK2 V617F and JAK1 V658F, and also provide new avenues to select for compounds able to prevent excessive IFNγ signaling, which is present in certain autoimmune conditions.Leroy, E.; et al. Biochem J2016,473 (11), 1579-91.Dusa, A.; et al. PLoS One2010,5 (6), e11157.Defour, J. P.; et al. Proc Natl Acad Sci U S A2013,110 (7), 2540-5.Constantinescu, S. N.; et al. Proc Natl Acad Sci U S A2001,98 (8), 4379-84.Lu, X.; et al. Proc Natl Acad Sci U S A2005,102 (52), 18962-7.Moraga, I.; et al. Cell2015,160 (6), 1196-208. DisclosuresConstantinescu:Novartis: Membership on an entity's Board of Directors or advisory committees; Personal Genetics: Membership on an entity's Board of Directors or advisory committees; MyeloPro Research and Diagnostics GmbH: Equity Ownership; Novartis: Consultancy; AlsaTECH: Equity Ownership; Novartis: Honoraria.

Mu Opioid Receptor Agonists with Dopamine D3 Receptor Antagonism: Toward Developing Analgesics with Less Abuse Potential

Opioid analgesics such as morphine are widely used in clinical practice for the management of moderate to severe pain. While they are the gold standard for painkillers, they are also highly addictive: approximately 10% of patients prescribed opioids for pain‐relief develop an opioid‐use disorder. Abuse of prescription opioids and heroin has become an epidemic in the United States with opioid overdose deaths quadrupling since 1999. Opioids induce their physiological effects through the activation of the mu opioid receptor (MOR). Interestingly, co‐administration of dopamine D3 (DRD3) receptor antagonists with opioids attenuates the self‐administration and rewarding effects of the drugs, but has no effect on their ability to relieve pain. This suggests that the development of compounds that both activate MOR and antagonize DRD3 may be a means to maintain the beneficial effects of opioids, while reducing their abuse potential. We have recently developed a library of small molecules with a novel scaffold that are agonists at MOR. Herein we screened our MOR agonists for antagonism at DRD3 in a DRD3/βarrestin enzyme fragment complementation assay. We identified multiple compounds that antagonize DRD3 that are as potent as Sulpiride, a known DRD3 antagonist. Further characterization of these MOR agonists with polypharmacology at DRD3 may allow for the development of opioid analgesics with reduced abuse liabilities.Support or Funding InformationFunded by NIDA grant R01 DA033073 to LMB Fellowship to NMS: NSF award 1359369. and TDB, and NSF Undergraduate ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.