Binding Assays Research Articles

Mutations in linker-2 of KLF1 impair expression of membrane transporters and cytoskeletal proteins causing hemolysis

The SP/KLF family of transcription factors harbour three C-terminal C2H2 zinc fingers interspersed by two linkers which confers DNA-binding to a 9-10 bp motif. Mutations in KLF1, the founding member of the family, are common. Missense mutations in linker two result in a mild phenotype. However, when co-inherited with loss-of-function mutations, they result in severe non-spherocytic hemolytic anemia. We generate a mouse model of this disease by crossing Klf1+/− mice with Klf1H350R/+ mice that harbour a missense mutation in linker-2. Klf1H350R/− mice exhibit severe hemolysis without thalassemia. RNA-seq demonstrate loss of expression of genes encoding transmembrane and cytoskeletal proteins, but not globins. ChIP-seq show no change in DNA-binding specificity, but a global reduction in affinity, which is confirmed using recombinant proteins and in vitro binding assays. This study provides new insights into how linker mutations in zinc finger transcription factors result in different phenotypes to those caused by loss-of-function mutations.

Long term investigation of formulation buffers to mitigate stability issues of conjugated critical reagents

Stability of conjugated critical reagents supporting ligand binding assays to enable biotherapeutic drug development is a universal concern. Formulation buffer employed for long-term cold storage may be key to mitigate protein aggregation issues. We investigated biophysical and functional attributes of murine mAb and human multispecific drug labeled with biotin, ruthenium, and Alexa fluor 647 frozen at −80 °C in PBS or a protein storage buffer for 3–15 months. Aggregation was observed at 4 months in mAb A-Ru (11.2%) and -Alexa (10%) in PBS followed by precipitation and reduced biological binding at 15 months. Increased aggregation in drug Ru (11.7%, 6 months) and Alexa (6.9%, 15 months) were noted but without impact on performance. There were no observations with biotin labeled reagents.

A transcriptional regulatory mechanism of genes in the tricarboxylic acid cycle in the heart

The tricarboxylic acid (TCA) cycle plays a crucial role in mitochondrial ATP production in the healthy heart. However, in heart failure, the TCA cycle becomes dysregulated. Understanding the mechanism by which TCA cycle genes are transcribed in the healthy heart is an important prerequisite to understanding how these genes become dysregulated in the failing heart. PGC-1α is a transcriptional coactivator that broadly induces genes involved in mitochondrial ATP production. PGC-1α potentiates its effects through coactivation of coupled transcription factors, such as ERR, Nrf1, Gabpa, and YY1. We hypothesized that PGC-1α plays an essential role in transcription of TCA cycle genes. Thus, by utilizing localization peaks of PGC-1α to TCA cycle gene promoters, it would allow the identification of coupled transcription factors. PGC-1α potentiated the transcription of 13 out of 14 TCA cycle genes, partly through ERR, Nrf1, Gabpa, and YY1. ChIP-sequencing showed PGC-1α localization peaks in TCA cycle gene promoters. Transcription factors with binding elements that were found proximal to PGC-1α peak localization were generally essential for transcription of the gene. These transcription factor binding elements were well conserved between mice and humans. Among the four transcription factors, ERR and Gabpa played a major role in potentiating transcription when compared to Nrf1 and YY1. These transcription factor-dependent PGC-1α recruitment was verified with Idh3a, Idh3g, and Sdha promoters with DNA binding assay. Taken together, this study clarifies the mechanism by which TCA cycle genes are transcribed, which could be useful to understand how those genes are dysregulated in pathological conditions.

Sub-10 μm Soft Interlayers Integrating Patterned Multivalent Biomolecular Binding Environments.

Designing surfaces that enable controlled presentation of multivalent ligand clusters (e.g., for rapid screening of biomolecular binding constants or design of artificial extracellular matrices) is a cross-cutting challenge in materials and interfacial chemistry. Existing approaches frequently rely on complex building blocks or scaffolds and are often specific to individual substrate chemistries. Thus, an interlayer chemistry that enabled efficient nanometer-scale patterning on a transferrable layer and subsequent integration with other classes of materials could substantially broaden the scope of surfaces available for sensors and wearable electronics. Recently, we have shown that it is possible to assemble nanometer-resolution chemical patterns on substrates including graphite, use diacetylene polymerization to lock the molecular pattern together, and then covalently transfer the pattern to amorphous materials (e.g., polydimethylsiloxane, PDMS), which would not natively enable high degrees of control over ligand presentation. Here, we develop a low-viscosity PDMS formulation that generates very thin films (<10 μm) with dense cross-linking, enabling high-efficiency surface functionalization with polydiacetylene arrays displaying carbohydrates and other functional groups (up to 10-fold greater than other soft materials we have used previously) on very thin films that can be integrated with other materials (e.g., glass and soft materials) to enable a highly controlled multivalent ligand display. We use swelling and other characterization methods to relate surface functionalization efficiency to the average distance between cross-links in the PDMS, developing design principles that can be used to create even thinner transfer layers. In the context of this work, we apply this approach using precision glycopolymers presenting structured arrays of N-acetyl glucosamine ligands for lectin binding assays. More broadly, this interlayer approach lays groundwork for designing surface layers for the presentation of ligand clusters on soft materials for applications including wearable electronics and artificial extracellular matrix.

Promotion of Raf-1/ASK1 complex formation by corylin inhibits cell apoptosis in myocardial ischemia/reperfusion injury

Effective treatment of myocardial ischemia–reperfusion (MIR) injury remains an unmet clinical need. Cardiomyocyte apoptosis is common at this stage and poses a significant risk. Corylin, a flavonoid compound extracted from Psoralea corylifolia L., has been shown to have anti-inflammatory, anticancer, and antiatherosclerotic properties. However, whether and how corylin affects MIR injury remain unclear. In this study, we explored the mechanism of corylin as a potent therapeutic agent for MI/R injury, using a left anterior descending (LAD) coronary artery ligation and oxygen–glucose deprivation and reperfusion (OGD/R) model in vivo and in vitro. TUNEL, Annexin-V/PI double staining,Ki67 immunohistochemistry, western blot analysis, and immunofluorescence were used to validate cell apoptosis level and Raf-1/ASK1 complex activity. The interaction between corylin and Raf-1/ASK1 complex was detected using molecular docking, corylin-Raf-1 binding assays, and coimmunoprecipitation (Co-IP). Moreover, TTC staining, echocardiography, HE staining, Masson trichrome staining and serological testing were performed to assess the cardioprotective effects of corylin in vivo. These findings showed that corylin reduces MIR injury-induced cardiomyocyte apoptosis and improves cardiac function. Mechanistically, corylin can interact with Raf-1 and promote the formation of the Raf-1/ASK1 complex, thus inhibiting cardiomyocyte apoptosis. In conclusion, our results demonstrate that corylin ameliorated cardiac dysfunction after MIR injury by reducing myocardial apoptosis.

η6-Arene Ru(II) Complexes as Modulators of Amyloid Aggregation.

Inorganic medicinal compounds represent a unique and versatile source of potential therapeutics in many diseases and, more recently, in neurodegeneration. Herein we investigated the effects of two η6-arene Ru(II) complexes on the self-aggregation processes of several amyloidogenic peptides endowed with different kinetics and primary sequences. The Ru(II) complexes exhibit, around the metal ion, two chlorides, one NHC = N-heterocyclic carbene, with a glucosyl and a methyl substituent and separately a hexamethylbenzene, which is named Ru1, and one benzene, named Ru2. Both complexes were demonstrated to bind monomeric amyloids suppressing aggregation as evidenced in thioflavin T (ThT) binding assays and autofluorescence experiments. Electrospray ionization mass spectrometry (ESI-MS) indicated the formation of direct adducts between amyloid and metal complexes, which determined the marked conformational variation of peptides and a rescue of cellular viability in SH-SY5Y cells. The complex Ru2 was demonstrated to be a more potent inhibitor of amyloid aggregation compared to Ru1 likely because of the less hindrance of the arene moiety. The presented data strongly support the in vitro ability of η6-arene Ru(II) complexes to suppress amyloid aggregation, providing insights into their potential application as novel therapeutics in neurodegenerative diseases.

One-pot synthesis of 1, 2-dihydro [1,2,4] triazinium salt by copperassisted unprecedented cyclization reaction: Application in protein binding.

Copper catalyzed intramolecular annulation of 2-((2benzylidene-1-phenylhydrazineyl)methyl)pyridine derivatives was described. It was found that Cu(II) is reduced under the reaction condition to Cu(I). Synthesized 1, 2-dihydro [1,2,4] triazinium salt showed fluorescence activity in solid state. On treating with base, an instant increase in fluorescence was observed. A detailed physicochemical assessment underscored the robust DNA-binding prowess of the [1,2,4] triazinium cationic species (C1-C3) via intercalative mechanisms. Notably, binding assays with BSA accentuated the heightened nucleic acid affinity of these cationic species.

Molecular basis for JAK2 inhibition with type II compounds.

176 Background: Upon prolonged exposure, the therapeutic efficiency of current clinical JAK2 inhibitors (type I inhibitors) can be attenuated, leading to drug resistance both in vitro and in vivo through non-genetic mechanisms. In contrast, type II inhibitors stabilize the inactive conformation of the kinase domain preventing the heterodimerization-mediated JAK2 activation. This confers efficacy to type I inhibitor persistent cells, improved efficacy against myeloproliferative neoplasms and other cancers such as B-cell acute lymphoblastic leukemia. Despite the advantages of type II JAK inhibitors, the discovery of type II inhibitor resistance mutation, L884P, in the kinase domain has hampered the further development of JAK2-targeted type II therapeutics. Methods: Here we use combination of protein-inhibitor binding assays, cell-based assays, X-ray crystallography, and computational tools to elucidate the resistance mechanism of JAK2-L884P mutation, to facilitate the development of type II inhibitors with activity against both wild-type and L884P mutated JAK2. Results: We have determined an atomic resolution crystal structure of JAK2-L884P mutant, revealing that mutation induces local changes in the structure but not in the ATP pocket. Furthermore, we have identified several type II compounds inhibiting. Interestingly, they show no major differences in potency in vitro against wild-type of L884P mutated JAK2. Selected compounds profiled in cell lines carrying wild-type or L884P-mutated JAK2, show differences in their cell viability, indicating that a resistance arises via allosteric effects. Conclusions: L884P mutation impacts the inhibitory potency of type II inhibitors via allosteric mechanism.Crystallographic analysis of our type II shortlisted compounds revealed that they differ in their binding modes and suggests that L884P-insensitive drugs can be developed. Preliminary cell-based experiments results confirm these findings. We will now focus on validating these observations with biomolecular simulations to clarify the molecular basis for the inhibitor resistance via L884P mutation. Our studies will facilitate the development of novel type II drugs against JAK2 in myeloproliferative neoplasms.

The surface protein GroEl of lactic acid bacteria mediates its modulation of the intestinal barrier in Penaeus vannamei

The molecular chaperone GroEL, commonly found in various bacterial species, exhibits heightened expression levels in response to high temperatures and increased levels of oxygen free radicals. Limited literature currently exists on the probiotic role of GroEL in invertebrates. This study sought to explore how the surface protein GroEL from Lactobacillus plantarum Ep-M17 impacts the intestinal barrier function of Penaeus vannamei. Through pull-down and immunofluorescence assays, the interaction between GroEL and Act1 in the gastrointestinal tract of P. vannamei was confirmed. Results from bacterial binding assays demonstrated that rGroEL can bind to pathogens like Vibrio parahaemolyticus E1 (V. p-E1). In vitro experiments revealed that the administration of rGroEL significantly decreased the levels of inflammatory cytokines induced by pathogens while preserving the integrity of tight junctions between intestinal epithelial cells and reducing bacteria-induced apoptosis. Additionally, rGroEL notably lessened the intestinal loading of V. p-E1 in P. vannamei, downregulated immune-related gene expression, and upregulated BCL/BAX expression in the intestines following V. p-E1 challenge. Mechanistic investigations further showed that rGroEL treatment effectively suppressed the expression and phosphorylation of proteins involved in the NF-κB and PI3K-AKT-mTOR signalling pathways in the intestines of bacteria-infected P. vannamei. Furthermore, GroEL reinforces protection against bacterial infections by enhancing the phagocytic and anti-apoptotic capabilities of P. vannamei hemocytes. These results suggest that GroEL may impede the interaction between pathogens and the intestinal mucosa through its competitive binding characteristics, ultimately reducing bacterial infections.

Neuropeptide Y Y2 Receptors in Sensory Neurons Tonically Suppress Nociception and Itch but Facilitate Postsurgical and Neuropathic Pain Hypersensitivity.

Neuropeptide Y (NPY) Y2 receptor (Y2) antagonist BIIE0246 can both inhibit and facilitate nociception. The authors hypothesized that Y2 function depends on inflammation or nerve injury status. The authors implemented a battery of behavioral tests in mice of both sexes that received (1) no injury; (2) an incision model of postoperative pain; (3) a spared nerve injury model of neuropathic pain; and (4) a latent sensitization model of chronic postsurgical pain. In addition to Y2 gene expression assays, spinal Y2 G-protein coupling was studied with guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assays. The authors report that intrathecal BIIE0246 increased mechanical and cold hypersensitivity, produced behavioral signs of spontaneous nociception and itch, and produced conditioned place aversion and preference in normal, uninjured mice. BIIE0246 did not change heat hypersensitivity or motor coordination. Conditional (sensory neuron-specific) Y2 deletion prevented BIIE0246-induced mechanical and cold hypersensitivity, nocifensive behaviors, and aversion. Both conditional deletion and pharmacologic blockade of Y2 reduced mechanical and thermal hypersensitivity after incision or nerve injury. SNI did not change the sensitivity of Y2 G-protein coupling with the Y2 agonist peptide YY (3-36) (PYY3-36), but increased the population of Y2 that effectively coupled G-proteins. Intrathecal PYY3-36 failed to reduce spared nerve injury- or incision-induced hypersensitivity in C57BL/6N mice. Incision did not change Npy2r gene expression in dorsal root ganglion. The authors conclude that Y2 at central terminals of primary afferent neurons provides tonic inhibition of mechanical and cold nociception and itch. This switches to the promotion of mechanical and thermal hyperalgesia in models of acute and chronic postsurgical and neuropathic pain, perhaps due to an increase in the population of Y2 that effectively couples to G-proteins. These results support the development of Y2 antagonists for the treatment of chronic postsurgical and neuropathic pain.

Receptor interactions of protoxin and activated Vip3Aa structural conformations in Spodoptera exigua.

The Vip3Aa insecticidal protein, produced by Bacillus thuringiensis, has been effectively used in commercial Bt-crops to manage lepidopteran pests. Upon ingestion by larvae, the protoxin is processed by midgut proteases into the activated protein and binds specifically to its receptors in the midgut, leading to insect mortality. Cryo-EM resolution of the trypsin-processed Vip3Aa protein unveiled structural remodelling of the N-terminal region during the transition from protoxin to activated protein. This conformational change has been demonstrated to be crucial for toxicity against Spodoptera exigua larvae, a major global lepidopteran pest. In this study, we investigated the relevance of the structural remodelling for the specific binding to midgut receptors. We conducted in vitro binding assays with radiolabelled proteins and brush border membrane vesicles (BBMV) from S. exigua, employing structural mutants that lock the protein in either its protoxin or its activated conformation. Our results indicate that both structural stages of the protein share binding sites in the midgut epithelium. Moreover, in vivo competition assays revealed that Vip3Aa is able to bind to functional receptors in S. exigua larvae both as protoxin and as activated protein. Altogether, our findings point to both structural conformations contributing to receptor binding. In vivo, either spontaneous structural shift upon proteolytic cleavage or receptor-mediated remodelling could be occurring. However, the timing and context in which the conformational change occurs could influence membrane insertion and toxicity. Our results show the complex interplay between proteolytic processing, protein structure and receptor interactions in Vip3Aa's toxicity. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Binding Interaction of Coumarin Derivative Daphnetin with Ovalbumin: Molecular Insights into the Complexation Process and Effects of Metal Ions and pH in the Binding and Antifibrillation Studies.

This study investigates the interaction between daphnetin and ovalbumin (OVA) as well as its potential to inhibit OVA fibrillation using both spectroscopic and computational analysis. A moderate binding affinity of 1 × 104 M-1 was observed between OVA and daphnetin, with a static quenched mechanism identified during the fluorescence quenching processes. Metal ions' (Cu2+ and Zn2+) presence led to an increase in the binding affinities of daphnetin toward OVA, mirroring a similar trend observed with the pH variation. Synchronous and 3D fluorescence studies indicated an increase in the polarity of the microenvironment surrounding the Trp residues during binding. Interestingly, circular dichroism and Fourier transform infrared studies showed a significant change in the secondary structure of OVA upon binding with daphnetin. The efficacy of daphnetin in inhibiting protein fibrillation was confirmed through thioflavin T and Congo Red binding assays along with fluorescence microscopic imaging analysis. The thermodynamic assessment showed positive ΔH° [+(29.34 ± 1.526) kJ mol-1] and ΔS° [+(181.726 ± 5.465) J mol-1] values, indicating the presence of the hydrophobic forces, while negative ΔG° signifies spontaneous binding interactions. These experimental findings were further correlated with computational analysis, revealing daphnetin dynamics within the binding site of OVA.

Sedum kamtschaticum Exerts Hypnotic Effects via the Adenosine A2A Receptor in Mice.

Insomnia is a common sleep disorder with significant societal and economic impacts. Current pharmacotherapies for insomnia are often accompanied by side effects, necessitating the development of new therapeutic drugs. In this study, the hypnotic effects and mechanisms of Sedum kamtschaticum 30% ethanol extract (ESK) and one of its active compounds, myricitrin, were investigated using pentobarbital-induced sleep experiments, immunohistochemistry (IHC), receptor binding assays, and enzyme-linked immunosorbent assay (ELISA). The pentobarbital-induced sleep experiments revealed that ESK and myricitrin reduced sleep latency and prolonged total sleep time in a dose-dependent manner. Based on c-Fos immunostaining, ESK, and myricitrin enhanced the GABAergic neural activity in sleep-promoting ventrolateral preoptic nucleus (VLPO) GABAergic. By measuring the level of GABA released from VLPO GABAergic neurons, ESK and myricitrin were found to increase GABA release in the hypothalamus. These effects were significantly inhibited by SCH. Moreover, ESK exhibited a concentration-dependent binding affinity for the adenosine A2A receptors (A2AR). In conclusion, ESK and myricitrin have hypnotic effects, and their underlying mechanisms may be related to the activation of A2AR.

MyACR: A Point-of-Care Medical Device for Determination of Albumin-Creatinine Ratio (uACR) in Random Urine Samples as a Marker of Nephropathy.

Chronic kidney disease (CKD) is a progressive condition that affects more than 10% of the world's population. Monitoring urine albumin-to-creatinine ratio (uACR) has become the gold standard for nephropathy diagnosis and control. The objective of the present study was to develop a simple, accurate, sensitive, and rapid point-of-care test (PoCT) device, MyACR, for uACR measurement, intended for use in community healthcare to screen for the risk and monitor the progress of CKD. Albumin and creatinine concentrations in urine samples were determined using spectrophotometric dye (tetrabromophenol blue)-binding and colorimetric Jaffe assay, respectively. Urine samples were diluted with distilled water (1:80) and mixed separately with albumin and creatinine reaction mixture. The creatinine reaction was incubated at room temperature (25 °C) for 30 min before analysis. Optical density (OD) was measured at the wavelengths of 625 nm (albumin) and 515 nm (creatinine). All calibration curves (0-60 mg/L and 0-2 mg/dL for albumin and creatinine) yielded linear relationships with correlation coefficients (R2) of >0.997. Good accuracy (% deviation of mean value (DMV) ≤ 5.42%) and precision (% coefficients of variation (CV) ≤ 12.69%) were observed from both the intra- and inter-day assays for the determination of albumin and creatinine using MyACR. The limit of quantification (LOQ) of albumin and creatinine in urine samples determined using MyACR and a laboratory spectrophotometer were 5 mg/L and 0.25 mg/dL, respectively, using 37.5 μL urine spiked samples (n = 5). The device was well-applied with clinical samples from 20 CKD patients. The median (range) of %DMV of the central (hospital) laboratory method (immune-based assay) was 3.48 (-17.05 to 21.64)%, with a high correlation coefficient (R2 > 0.98). In conclusion, MyACR showed satisfactory test performance in terms of accuracy, reproducibility, and sensitivity. Cost-effectiveness and improvement in clinical decision making need to be proven in future multisite community and home studies.

Structural Characterization of Human Bufavirus 1: Receptor Binding and Endosomal pH-Induced Changes.

Bufaviruses (BuV) are members of the Parvoviridae of the Protoparvovirus genus. They are non-enveloped, T = 1 icosahedral ssDNA viruses isolated from patients exhibiting acute diarrhea. The lack of treatment options and a limited understanding of their disease mechanisms require studying these viruses on a molecular and structural level. In the present study, we utilize glycan arrays and cell binding assays to demonstrate that BuV1 capsid binds terminal sialic acid (SIA) glycans. Furthermore, using cryo-electron microscopy (cryo-EM), SIA is shown to bind on the 2/5-fold wall of the capsid surface. Interestingly, the capsid residues stabilizing SIA binding are conserved in all human BuVs identified to date. Additionally, biophysical assays illustrate BuV1 capsid stabilization during endo-lysosomal (pH 7.4-pH 4) trafficking and capsid destabilization at pH 3 and less, which correspond to the pH of the stomach. Hence, we determined the cryo-EM structures of BuV1 capsids at pH 7.4, 4.0, and 2.6 to 2.8 Å, 3.2 Å, and 2.7 Å, respectively. These structures reveal capsid structural rearrangements during endo-lysosomal escape and provide a potential mechanism for this process. The structural insights gained from this study will add to the general knowledge of human pathogenic parvoviruses. Furthermore, the identification of the conserved SIA receptor binding site among BuVs provides a possible targetable surface-accessible pocket for the design of small molecules to be developed as anti-virals for these viruses.

The process using a synthetic library that generates multiple diverse human single domain antibodies.

Single domain antibodies (sdAbs) possess unique characteristics that make them highly effective for developing complex therapeutics. Our process uses a fully synthetic phage display library to generate single domain antibodies that can bind to disease relevant antigen conformations. A human IGHV3 family scaffold makes up the phage display libraries, and these VHO libraries are applied to diverse phage biopannings against target antigens. After NGS processing, unique VHOs undergo automated cloning into expression constructs followed by transfections and purifications. Binding assays were used to determine VHO binding behaviors to the target proteins. Additional VHO interactions are measured against endogenous targets on cells by way of flow cytometry, cell internalization, and activation assays. We show that a fully synthetic phage display library can generate VHOs that bind to disease relevant antigen conformations. The diverse biopanning methods and processing of next-generation sequencing generated many VHO paratopes. These different VHO sequences can be expressed as Fc fusion proteins. Various screening assays resulted in VHOs representing different epitopes or activities. During the hit evaluation, we demonstrate how screening can identify distinct VHO activities that have been used to generate differentiated drug molecules in various bispecific and multispecific antibody formats. We demonstrate how screening can identify distinct VHO activities that have been used to generate differentiated drug molecules in various bispecific and multispecific antibody formats.

PET imaging of colon cancer CD73 expression using cysteine site-specific 89Zr-labeled anti-CD73 antibody

CD73 is a cell-surface ectoenzyme that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine, which in turn can promote resistance to immune checkpoint blockade therapy. Immune response may therefore be improved by targeting tumor CD73, and this possibility underlines the need to non-invasively assess tumor CD73 level. In this study, we developed a cysteine site-specific 89Zr-labeled anti-CD73 (89Zr-CD73) IgG immuno-PET technique that can image tumor CD73 expression in living bodies. Anti-CD73 IgG was reduced with tris(2-carboxyethyl)phosphine, underwent sulfohydryl moiety–specific conjugation with deferoxamine–maleimide, and was radiolabeled with 89Zr. CT26 mouse colon cancer cells, CT26/CD73 cells engineered to constitutively overexpress CD73, and 4T1.2 mouse breast cancer cells underwent cell binding assays and western blotting. Balb/c nude mice bearing tumors underwent 89Zr-CD73 IgG PET imaging and biodistribution studies. 89Zr-CD73 IgG showed 20-fold higher binding to overexpressing CT26/CD73 cells compared to low-expressing CT26 cells, and moderate expressing 4T1.2 cells showed uptake that was 38.9 ± 1.51% of CT26/CD73 cells. Uptake was dramatically suppressed by excess unlabeled antibody. CD73 content proportionately increased in CT26 and CT26/CD73 cell mixtures was associated with linear increases in 89Zr-CD73 IgG uptake. 89Zr-CD73 IgG PET/CT displayed clear accumulation in CT26/CD73 tumors with greater uptake compared to CT26 tumors (3.13 ± 1.70%ID/g vs. 1.27 ± 0.31%ID/g at 8 days; P = 0.04). Specificity was further supported by low CT26/CD73 tumor-to-blood ratio of 89Zr-isotype-IgG compared to 89Zr-CD73 IgG (0.48 ± 0.08 vs. 2.68 ± 0.52 at 4 days and 0.53 ± 0.07 vs. 4.81 ± 1.02 at 8 days; both P < 0.001). Immunoblotting and immunohistochemistry confirmed strong CD73 expression in CT26/CD73 tumors and low expression in CT26 tumors. 4T1.2 tumor mice also showed clear 89Zr-CD73 IgG accumulation at 8 days (3.75 ± 0.70%ID/g) with high tumor-to-blood ratio compared to 89Zr-isotype-IgG (4.91 ± 1.74 vs. 1.20 ± 0.28; P < 0.005). 89Zr-CD73 IgG specifically targeted CD73 on high expressing cancer cells in vitro and tumors in vivo. Thus, 89Zr-CD73 IgG immuno-PET may be useful for the non-invasive monitoring of CD73 expression in tumors of living subjects.

Interleukin-1β Signaling Contributes to Cell Cycle Arrest and Apoptotic Cell Death by Leptin via Modulation of AKT and p38MAPK in Hepatocytes.

Leptin, an adipose tissue-derived hormone, has exhibited the potent hepatotoxic effects. However, the underlying molecular mechanisms are not fully understood. In this study, we have elucidated the mechanisms by which leptin exerts cytotoxic effects in hepatocytes, particularly focusing on the role of interleukin-1β (IL-1β) signaling. Leptin significantly induced maturation and secretion of IL-1β in cultured rat hepatocytes. Interestingly, inhibition of IL-1β signaling by pretreatment with an IL-1 receptor antagonist (IL-1Ra) or gene silencing of type I IL-1 receptor (IL-1R1) markedly abrogated leptin-induced cell cycle arrest. The critical role of IL-1β signaling in leptin-induced cell cycle arrest is mediated via upregulation of p16, which acts as an inhibitor of cyclin-dependent kinase. In addition, leptin-induced apoptotic cell death was relieved by inhibition of IL-1β signaling, as determined by annexin V/7-AAD binding assay. Mechanistically, IL-1β signaling contributes to apoptotic cell death and cell cycle arrest by suppressing AKT and activation of p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Involvement of IL-1β signaling in cytotoxic effect of leptin was further confirmed in vivo using hepatocyte specific IL-1R1 knock out (IL-1R1 KO) mice. Essentially similar results were obtained in vivo, where leptin administration caused the upregulation of apoptotic markers, dephosphorylation of AKT, and p38MAPK activation were observed in wild type mice liver without significant effects in the livers of IL-1R1 KO mice. Taken together, these results demonstrate that IL-1β signaling critically contributes to leptin-induced cell cycle arrest and apoptosis, at least in part, by modulating p38MAPK and AKT signaling pathways.

Abstract Tu109: Sirt1 inhibits the Sub1-induced RNA polymerase II recruitment to metabolic gene promoters during pressure overload

Downregulation of genes involved in mitochondrial energy production is a hallmark of heart failure, which associates with inhibition of RNA polymerase II (Pol II) recruitment to the gene promoters. Sirt1 is a protein deacetylase regulating transcription of metabolic genes. However, how Sirt1 regulates metabolic genes in heart failure is controversial. Here we show that Sirt1 promotes pressure overload (PO)-induced downregulation of metabolic genes possibly through inhibition of the Pol II recruitment. Cardiac-specific Sirt1 overexpression in mice (Tg-Sirt1) promoted PO-induced cardiac dysfunction (Ejection fraction (%): wild type (WT) sham 76, WT PO 68, Tg-Sirt1 sham 76, Tg-Sirt1 PO 48*, p&lt;0.05 vs WT PO) and downregulation of metabolic genes, including Idh3a (relative Idh3a mRNA levels; WT Sham 1.0, WT PO 0.55, Tg-Sirt1 0.98, Tg-Sirt1 PO 0.41, p&lt;0.05 vs WT PO). In cultured cardiomyocytes, Sirt1 overexpression suppressed metabolic enzymes, reporter gene activity driven by endogenous metabolic gene promoters, and mitochondrial respiration. Chromatin immunoprecipitation and in vitro DNA binding assays, using 500bp of endogenous Idh3a promoter sequence, showed that Sirt1 inhibits the Pol II recruitment to the promoter. Unbiased screening using mass spectrometry identified Sub1 as a Sirt1 binding protein. Sub1 is an evolutionally conserved DNA binding protein that mediates the Pol II recruitment via the preinitiation complex formation. We confirmed direct binding of Sub1 to the Sirt1 catalytic domain (235-499aa) and Sub1-dependent Sirt1 recruitment to the Idh3a promoter using the in vitro reconstitution system. In vitro DNA binding assays with the Idh3a promoter sequence demonstrated that Sirt1 inhibits preinitiation complex formation, but not Sub1 recruitment. These results suggest that Sirt1 inhibits Sub1-induced preinitiation complex formation, thereby suppressing transcription of metabolic genes in the failing heart.

Abstract We137: PGC-1α-dependent transcriptional regulatory mechanisms for tricarboxylic acid cycle genes in the heart.

Many metabolic genes, including tricarboxylic acid (TCA) cycle genes, are downregulated in heart failure, which may promote the pathology due to insufficient energy production. To clarify how TCA cycle genes are downregulated in the failing heart, it is a prerequisite to clarify how these genes are transcribed in the normal heart. PGC-1α is a transcriptional co-activator that broadly induces metabolic genes through activation of transcription factors, such as ERR, Nrf1, Gabpa and YY1. Here we show that PGC-1α transcribes 13 among 14 TCA cycle genes partly through ERR, Nrf1, Gabpa and YY1. Thirteen TCA cycle genes, except for Suclg2, were downregulated in cardiac specific PGC-1α knockout (KO) mice (Relative mRNA levels in KO mice compared to wild type (WT) were CS 0.66*, Aco2 0.64*, Idh3a 0.62*, Idh3b 0.54*, Idh3g 0.54*, Ogdh 0.64*, Suclg1 0.45*, Suclg2: 1.1, Sdha 0.57*, Sdhb 0.53*, Sdhc 0.72*, Sdhd 0.57*, Fh1 0.32* and Mdh2 0.54*,p&lt;0.05 vs. WT). ChIP-sequencing with PGC-1α antibody demonstrated that PGC-1α is localized at the promoter of thirteen TCA cycle genes, besides Suclg2. Bioinformatics analyses identified consensus binding sequences of ERR, Nrf1, Gabpa and YY1 in these promoters. Gene expression analyses with knockdown of these transcription factors demonstrated that a transcription factor whose binding sequence is found proximal to the PGC-1α localization peak transcribes the downstream genes in general. These transcription factor binding sequences were well conserved between mice and humans. ERR-, Nrf1- and Gabpa-dependent PGC-1α recruitment was verified with in vitro DNA binding assays using endogenous promoter sequences of Idh3a, Idh3g and Sdha. Taken together, this study clarifies a precise transcriptional mechanism for TCA cycle genes in the normal heart, which could be useful for understanding how those genes are dysregulated in the failing heart.