Ligand Binding Activity Research Articles

Notch1 blockade by a novel, selective anti-Notch1 neutralizing antibody improves immunotherapy efficacy in melanoma by promoting an inflamed TME

BackgroundImmune checkpoint inhibitors (ICI) have dramatically improved the life expectancy of patients with metastatic melanoma. However, about half of the patient population still present resistance to these treatments. We have previously shown Notch1 contributes to a non-inflamed TME in melanoma that reduces the response to ICI. Here, we addressed the therapeutic effects of a novel anti-Notch1 neutralizing antibody we produced, alone and in combination with immune checkpoint inhibition in melanoma models.MethodsAnti-Notch1 was designed to interfere with ligand binding. Mice were immunized with a peptide encompassing EGF-like repeats 11–15 of human Notch1, the minimal required region that allows ligand binding and Notch1 activation. Positive clones were expanded and tested for neutralizing capabilities. Anti-Notch1-NIC was used to determine whether anti-Notch1 was able to reduce Notch1 cleavage; while anti-SNAP23 and BCAT2 were used as downstream Notch1 and Notch2 targets, respectively. K457 human melanoma cells and the YUMM2.1 and 1.7 syngeneic mouse melanoma cells were used. Cell death after anti-Notch1 treatment was determined by trypan blue staining and compared to the effects of the gamma-secretase inhibitor DBZ. 10 mg/kg anti-Notch1 was used for in vivo tumor growth of YUMM2.1 and 1.7 cells. Tumors were measured and processed for flow cytometry using antibodies against major immune cell populations.ResultsAnti-Notch1 selectively inhibited Notch1 but not Notch2; caused significant melanoma cell death in vitro but did not affect normal melanocytes. In vivo, it delayed tumor growth without evident signs of gastro-intestinal toxicities; and importantly promoted an inflamed TME by increasing the cytotoxic CD8+ T cells while reducing the tolerogenic Tregs and MDSCs, resulting in enhanced efficacy of anti-PD-1.ConclusionsAnti-Notch1 safely exerts anti-melanoma effects and improves immune checkpoint inhibitor efficacy. Thus, anti-Notch1 could represent a novel addition to the immunotherapy repertoire for melanoma.

Identification and Activity Study of an Impurity Band Observed in the nrSDS-PAGE of Aflibercept.

Aflibercept is a biopharmaceutical targeting vascular endothelial growth factor (VEGF) that has shown promise in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) in adults. Quality control studies of aflibercept employing non-reduced SDS-PAGE (nrSDS-PAGE) have shown that a significant variant band (IM1) is consistently present below the main band. Considering the quality control strategy of biopharmaceuticals, structural elucidation and functional studies are required. In this study, the variant bands in nrSDS-PAGE were collected through electroelution and identified by peptide mass fingerprinting based on liquid chromatography-tandem MS (LC-MS/MS). This variant was expressed using knob-into-hole (KIH) design transient transfection for the detection of ligand affinity, binding activity and biological activity. The variant band was formed by C-terminal truncation at position N99 of one chain in the aflibercept homodimer. Then, this variant was successfully expressed using KIH design transient transfection. The ligand affinity of the IM1 truncated variant was reduced by 18-fold, and neither binding activity nor biological activity were detected. The efficacy of aflibercept is influenced by the loss of biological activity of the variant. Therefore, this study supports the development of a quality control strategy for aflibercept.

Multigenerational effects of disperse blue 79 at environmentally relevant concentrations on zebrafish (Danio rerio) fecundity: An integrated approach

The brominated azo dye (BAD) Disperse Blue (DB79) is a widespread environmental pollutant. The long-term toxicological effects of DB79 and the mechanisms thereof must be understood to allow assessment of the risks of DB79 pollution. A dual-omics approach employing in silico analysis, bioinformatics, and in vitro bioassays was used to investigate the transgenerational (F0–F2) toxicity of DB79 in zebrafish at environmentally relevant concentrations and identify molecular initiating events and key events associated with DB79-induced fertility disorders. Exposure to 500 µg/L DB79 decreased fecundity in the F0 and F1 generations by > 30 % and increased the condition factor of the F1 generation 1.24-fold. PPARα/RXR and PXR ligand binding activation were found to be critical molecular initiating events associated with the decrease in fecundity. Several key events (changes in fatty acid oxidation and uptake, lipoprotein metabolism, and xenobiotic metabolism and transport) involved in lipid dysregulation and xenobiotic disposition were found to be induced by DB79 through bioinformatic annotation using dual-omics data. The biomolecular underpinnings of decreased transgenerational fertility in zebrafish attributable to BAD exposure were elucidated and novel biomolecular targets in the adverse outcome pathway framework were identified. These results will inform future studies and facilitate the development of mitigation strategies.

Bivalent affinity binding-inspired PPARγ immobilization with selective conformation and improved ligand-binding activity

Functional protein immobilization forms the basis for bio-detections. A series of one-point, site-specific immobilization methods have been developed, however, it still remains as a challenge how to avoid the proteins to move in all directions as well as conveniently regenerate the bio-devices. Herein, we have developed a bivalent affinity binding-inspired method for PPARγ immobilization using DNA aptamer and nickel-nitrilotriacetic acid (Ni2+-NTA) chelation. The specific DNA aptamer (Apt 2) was selected by an on-column systematic evolution of ligands by exponential enrichment (SELEX) method with affinity of (1.57 ± 0.15) × 105 M−1, determined by isothermal titration calorimetry (ITC). Apt 2 and nickel-nitrilotriacetic acid (Ni2+-NTA) were modified on macroporous silica gels via L-α-allylglycine as a linker. They respectively interacted with PPARγ and 6×His tag via bivalent affinity binding for the receptor immobilization. After comprehensive surface characterization, PPARγ was proved to be successful immobilized. Chromatographic studies revealed that the immobilized PPARγ has conformation selectivity, which discriminated agonist and antagonist of the receptor. Ligand-binding parameters (affinity and rate constant) of four agonists (rosiglitazone, pioglitazone, troglitazone, and magnolol) with PPARγ were determined. Troglitazone showed the lowest dissociation rate constant. The binding affinities (3.28 × 107, 1.91 × 106, 2.25 × 107, and 2.43 × 107 M−1) were highly consistent with the data obtained using purified receptor in solution (2.16 × 107, 4.52 × 106, 1.20 × 107, and 1.56 × 107 M−1), offering reliable bio-detection method for PPARγ and its ligands. Due to the biocompatibility of nuclear receptor with DNA, it is conceivable that the bivalent affinity-based method will be a general method for the immobilization of other nuclear receptors, which may provide selective conformation and improved ligand-binding activity for the receptors.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

Heme pocket modulates protein conformation and diguanylate cyclase activity of a tetrameric globin coupled sensor

Bacteria use the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) to control biofilm formation and other key phenotypes in response to environmental signals. Changes in oxygen levels can alter c-di-GMP signaling through a family of proteins termed globin coupled sensors (GCS) that contain diguanylate cyclase domains. Previous studies have found that GCS diguanylate cyclase activity is controlled by ligand binding to the heme within the globin domain, with oxygen binding resulting in the greatest increase in catalytic activity. Herein, we present evidence that heme-edge residues control O2-dependent signaling in PccGCS, a GCS protein from Pectobacterium carotovorum, by modulating heme distortion. Using enzyme kinetics, resonance Raman spectroscopy, small angle X-ray scattering, and multi-wavelength analytical ultracentrifugation, we have developed an integrated model of the full-length PccGCS tetramer and have identified conformational changes associated with ligand binding, heme conformation, and cyclase activity. Taken together, these studies provide new insights into the mechanism by which O2 binding modulates activity of diguanylate cyclase-containing GCS proteins.

Structural mechanisms for binding and activation of a contact-quenched fluorophore by RhoBAST.

The fluorescent light-up aptamer RhoBAST, which binds and activates the fluorophore-quencher conjugate tetramethylrhodamine-dinitroaniline with high affinity, super high brightness, remarkable photostability, and fast exchange kinetics, exhibits excellent performance in super-resolution RNA imaging. Here we determine the co-crystal structure of RhoBAST in complex with tetramethylrhodamine-dinitroaniline to elucidate the molecular basis for ligand binding and fluorescence activation. The structure exhibits an asymmetric "A"-like architecture for RhoBAST with a semi-open binding pocket harboring the xanthene of tetramethylrhodamine at the tip, while the dinitroaniline quencher stacks over the phenyl of tetramethylrhodamine instead of being fully released. Molecular dynamics simulations show highly heterogeneous conformational ensembles with the contact-but-unstacked fluorophore-quencher conformation for both free and bound tetramethylrhodamine-dinitroaniline being predominant. The simulations also show that, upon RNA binding, the fraction of xanthene-dinitroaniline stacked conformation significantly decreases in free tetramethylrhodamine-dinitroaniline. This highlights the importance of releasing dinitroaniline from xanthene tetramethylrhodamine to unquench the RhoBAST-tetramethylrhodamine-dinitroaniline complex. Using SAXS and ITC, we characterized the magnesium dependency of the folding and binding mode of RhoBAST in solution and indicated its strong structural robustness. The structures and binding modes of relevant fluorescent light-up aptamers are compared, providing mechanistic insights for rational design and optimization of this important fluorescent light-up aptamer-ligand system.

Evaluation of the Homogenous Mobility Shift Assay for Infliximab and Adalimumab Anti-drug Antibody Detection in the Clinical Laboratory.

Detecting antidrug antibodies (ADAs) against infliximab or adalimumab is useful for therapeutic drug monitoring. Various ADA detection methods exist, and antibody titer is an output in some algorithms. Homogenous mobility shift assay (HMSA) measures relative ADA concentration and determines drug-ADA complex size in vitro. However, the relevance of complex size determination in drug monitoring remains unclear. Hence, the association between complex size, ADA concentration, and sample detectable neutralizing activity was evaluated. Sera from infliximab-treated and adalimumab-treated patients who tested positive for ADA in the National Screening Service were analyzed using 3 ADA assays. HMSA determined the relative ADA concentrations and complex sizes, competitive ligand-binding assay evaluated the sample neutralizing capacity, and enzyme-linked immunosorbent assay detected immunoglobulin (Ig)G4 ADA. Most ADA-positive samples (>80%) formed drug-ADA dimer complexes, whereas 17% had dimer and multimer complexes, and 3% had multimeric complexes. Multimer presence had 100% positive predictive value for detectable neutralizing activity. ADA concentration and detectable neutralizing activity were moderately correlated (r = 0.65) in adalimumab-treated patients and strongly correlated (r = 0.81) in infliximab-treated patients. In adalimumab-treated patients, multimer presence was a stronger predictor of neutralizing activity than ADA concentration was, but not in infliximab-treated patients. However, in infliximab-treated patient samples, multimer presence revealed a distinct subset with high ADA concentrations, neutralizing activity, and IgG4 ADA. Multimers detected using HMSA had a strong positive predictive value for competitive ligand-binding assay detectable neutralizing activity. Multimeric IgG4-containing ADA-drug complexes revealed a distinct subset of infliximab-treated patient samples, whose clinical relevance merits further investigation.

Investigating the effect of SUMO fusion on solubility and stability of amylase-catalytic domain from Pyrococcus abyssi

Alpha amylase belonging to starch hydrolyzing enzymes has significant contributions to different industrial processes. The enzyme production through recombinant DNA technology faces certain challenges related to their expression, solubility and purification, which can be overcome through fusion tags. This study explored the influence of SUMO, a protein tag reported to enhance the solubility and stability of target proteins when fused to the N-terminal of the catalytic domain of amylase from Pyrococcus abyssi (PaAD). The insoluble expression of PaAD in E. coli was overcome when the enzyme was expressed in a fusion state (S-PaAD) and culture was cultivated at 18 °C. Moreover, the activity of S-PaAD increased by 1.5-fold as compared to that of PaAD. The ligand binding and enzyme activity assays against different substrates demonstrated that it was more active against 1 % glycogen and amylopectin. The analysis of the hydrolysates through HPLC demonstrated that the enzyme activity is mainly amylolytic, producing longer oligosaccharides as the major end product. The secondary structure analyses by temperature ramping in CD spectroscopy and MD simulation demonstrated the enzymes in the free, as well as fusion state, were stable at 90 °C. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various foods, feed, textiles, detergents, pharmaceuticals, and many industrial applications.

An odorant-binding protein based electrical sensor to detect volatile organic compounds

Artificial olfaction systems, such as electronic nose devices (ENs), can be used in various fields, from healthcare to food industry and the environmental sector. ENs consist of an array of sensors composed of different sensing materials. Incorporating Odorant Binding Proteins (OBPs) into gas-sensing materials can increase volatile organic compounds (VOCs) recognition and selectivity. OBPs are small soluble proteins found in vertebrates and insects, responsible for VOCs solubilization and transportation. In this work, OBP3 from Rattus norvegicus was selected to develop an OBP-based electrical VOC sensor, by optimizing protein production and immobilization on sensors surface. OBP3 was successfully produced in E. coli host cells (94 mg/L) and purified with high purity (88% purification yield, 96% purity). The protein folding and thermal stability were assessed by circular dichroism (Tm=71±1ºC) whereas ligand binding activity was verified in solution by fluorescence displacement against diisopropylphenol (Kd=0.24 µM). For the immobilization of OBP3 on gold interdigitated electrodes modified with reduced graphene oxide, we explored two strategies (covalent and non-covalent), establishing a reproducible and cost-effective methodology to develop OBP3-based electrical sensors. The non-covalent immobilization of a linker to the graphene-modified surface showed improved outcomes compared to the carbodiimide crosslinking chemistry. OBP3-sensors presented selectivity towards distinct model compounds in the gas phase (diisopropylphenol, dimethylpyrazine, menthone and decanol), in correlation with the dissociation constants measured by fluorescence displacement assays in solution. As a result, this study expands the practical applications of OBPs for gas-phase sensors, showcasing their potential for enhancing VOC detection.

Bioinformatics analysis of missense mutations in CXCR1 implicates altered protein stability and function

Human CXCR1 is a G-protein α subunit i (Gαi)-coupled receptor (GPCR) that plays an important role in promoting leukocyte recruitment and activation in inflammatory regions; thus, its genetic contribution to human disorders warrants further investigation. In this study, we investigated whether oncogenic missense mutations in CXCR1 would affect its activity and hinder its ability to interact with its ligand. This study utilized a bioinformatics approach and employed precise and thorough computational methods to gain insights into the molecular characteristics of mutated CXCR1 that are responsible for causing diseases. I-TASSER was used to construct a mutant model with the required mutations. Schrödinger’s Desmond software was used to evaluate how mutations affect the stability and function of proteins. In this study, 299 CXCR1 missense mutations were examined; 53 of these were reported to be disease-causing, five of which were directly associated with cancer. The impact of the three cancer-causing mutations (N57D, R135C, and P302S) on protein stability and function was subsequently examined through computational analysis. Positions N57, R135, and P302 were determined to be highly conserved, and substitutions with aspartic acid (D), cysteine (C), and serine (S), respectively, could impair CXCR1 activity. Hence, our findings suggested that these mutations could alter CXCR1 ligand binding activity, lowering the risk of cancer and helping patients defend against pathogen invasion during a neutrophil-mediated innate immune response.

Scaling up Functional Analyses of the G Protein-Coupled Receptor Rhodopsin.

Eukaryotic cells use G protein-coupled receptors (GPCRs) to convert external stimuli into internal signals to elicit cellular responses. However, how mutations in GPCR-coding genes affect GPCR activation and downstream signaling pathways remain poorly understood. Approaches such as deep mutational scanning show promise in investigations of GPCRs, but a high-throughput method to measure rhodopsin activation has yet to be achieved. Here, we scale up a fluorescent reporter assay in budding yeast that we engineered to study rhodopsin's light-activated signal transduction. Using this approach, we measured the mutational effects of over 1200 individual human rhodopsin mutants, generated by low-frequency random mutagenesis of the GPCR rhodopsin (RHO) gene. Analysis of the data in the context of rhodopsin's three-dimensional structure reveals that transmembrane helices are generally less tolerant to mutations compared to flanking helices that face the lipid bilayer, which suggest that mutational tolerance is contingent on both the local environment surrounding specific residues and the specific position of these residues in the protein structure. Comparison of functional scores from our screen to clinically identified rhodopsin disease variants found many pathogenic mutants to be loss of function. Lastly, functional scores from our assay were consistent with a complex counterion mechanism involved in ligand-binding and rhodopsin activation. Our results demonstrate that deep mutational scanning is possible for rhodopsin activation and can be an effective method for revealing properties of mutational tolerance that may be generalizable to other transmembrane proteins.

Formation of EGFRwt/EGFRvIII homo- and hetero-dimers in glioblastoma cells as detected by single molecule localization microscopy.

Super-resolution microscopy has been used to show the formation of receptor clusters and adapted lipid organization of cell membranes for many members of the ErbB receptor family. The clustering behaviour depends on the receptor size and shape, possibly ligand binding or expression activity. Using single molecule localization microscopy (SMLM), we also showed this typical clustering for the epidermal growth factor receptor variant III (EGFRvIII) in glioblastoma multiforme (GBM) cells. EGFRvIII is co-expressed with the wild type (EGFRwt) and both receptors are assumed to preferentially form hetero-dimers leading to transactivation and elevated oncogenic EGFR-signalling in GBM cells. Here, we analysed EGFRvIII and EGFRwt co-localization using our already described model system of the glioblastoma cell line DKMG, displaying endogenous EGFRvIII expression. Using EGFRvIII and EGFRwt specific antibodies, EGFR localization and their potential for dimerization in a given membrane cluster were analysed by dual colour SMLM supported by novel approaches of mathematic evaluations including Ripley statistics, persistent homology and similarity algorithms. Surprisingly, cluster analysis, Ripley point-to-point distance statistics for cluster geometry and persistent homology comparing cluster topology, revealed that both EGFRvIII and EGFRwt do primarily not form hetero-dimers but the results support the hypothesis that they tend to form homo-dimers. The ratio of homo-dimers obtained by this calculation was significantly higher (>5σ, standard deviation) than expected from randomly arranged points. In comparison, hetero-dimer formation was only slightly increased. We confirmed these data by immunoprecipitation, which show no co-precipitation of EGFRvIII and EGFRwt. Furthermore, we showed that the topology of the clusters was more similar among the same type than among the different types of receptors. Taken together, these data indicate that EGFRvIII does induce oncogenic signalling by homo-dimerisation and not preferentially by hetero-dimer formation with EGFRwt. These data offer a new perspective on EGFRvIII signalling which will lead to a better understanding of this tumour associated receptor variant in GBM.

Structural Insights into the Activation of Human Aryl Hydrocarbon Receptor by the Environmental Contaminant Benzo[a]pyrene and Structurally Related Compounds

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor belonging to the bHLH/PAS protein family and responding to hundreds of natural and chemical substances. It is primarily involved in the defense against chemical insults and bacterial infections or in the adaptive immune response, but also in the development of pathological conditions ranging from inflammatory to neoplastic disorders. Despite its prominent roles in many (patho)physiological processes, the lack of high-resolution structural data has precluded for thirty years an in-depth understanding of the structural mechanisms underlying ligand-binding specificity, promiscuity and activation of AHR. We recently reported a cryogenic electron microscopy (cryo-EM) structure of human AHR bound to the natural ligand indirubin, the chaperone Hsp90 and the co-chaperone XAP2 that provided the first experimental visualization of its ligand-binding PAS-B domain. Here, we report a 2.75 Å resolution structure of the AHR complex bound to the environmental pollutant benzo[a]pyrene (B[a]P). The structure substantiates the existence of a bipartite PAS-B ligand-binding pocket with a geometrically constrained primary binding site controlling ligand binding specificity and affinity, and a secondary binding site contributing to the binding promiscuity of AHR. We also report a docking study of B[a]P congeners that validates the B[a]P-bound PAS-B structure as a suitable model for accurate computational ligand binding assessment. Finally, comparison of our agonist-bound complex with the recently reported structures of mouse and fruit fly AHR PAS-B in different activation states suggests a ligand-induced loop conformational change potentially involved in the regulation of AHR function.

Abstract C098: Targeting high-expression LGR5 by an R-spondin-based peptibody-drug conjugate is efficacious against neuroblastoma

Abstract Neuroblastoma (NB) is a pediatric tumor that predominantly affects infants and young children, with high-risk (HR) disease being diagnosed in approximately 50% of patients at 18 months or older. LGR4-6 are membrane receptors that bind RSPO ligands, activating Wnt signaling. LGR5, specifically expressed in adult stem cells, is highly upregulated in gastrointestinal (GI) cancers. In NB, high LGR5 expression is associated with aggressive disease and poor outcomes. Knockdown of LGR5 reduces cell growth in LGR5-high NB cell lines. The R-spondin-LGR5 axis has emerged as a potential therapeutic target due to its involvement in stem cell proliferation and self-renewal, as well as its overexpression in certain cancers, including neuroblastoma. RSPO-based peptibody drug conjugate (PDC) is a promising approach to target LGR4/5 for HR-NB therapy. PDCs are designed to selectively deliver the potent Pyrrolobenzodiazepine (PBD) dimer SG3199 to LGR5 high-expressing neuroblastoma cells using a peptibody that mimics the R-spondin binding activity of LGR5 ligands. Our preclinical study showed the efficacy of DAR2 R-spondin-based PDCs, generated through microbial transglutaminase-mediated site-specific conjugation, inducing cell death both in vitro and in vivo. Furthermore, these PDCs demonstrate reduced tumor growth and improved survival in xenograft animal models of neuroblastoma derived from the SK-N-AS cell lines. Our findings suggest that targeting LGR5 with RSPO2-based PDCs may offer a promising potential for treating neuroblastoma. Citation Format: Yukimatsu Toh. Targeting high-expression LGR5 by an R-spondin-based peptibody-drug conjugate is efficacious against neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C098.

Evaluation of Direct Ligand-Receptor Interactions by Photoaffinity Labeling.

Binding assays provide ultimate proof that a particular peptide and receptor kinase (RK) do indeed function as a ligand-receptor pair. Among available binding assays, proximity-induced photoaffinity labeling is superior for confirming direct contact between the peptide ligand and the receptor. Our binding assay employs covalent photoaffinity labeling followed by immunoprecipitation to specifically evaluate the ligand binding activity of the target RKs. Here, we describe a protocol for the synthesis of photoactivatable peptide ligands and the UV-induced formation of covalent bonds between photoaffinity ligands and RKs.

Functional Characterization of Novel MC4R Variants Identified in Two Unrelated Patients with Morbid Obesity in Qatar.

The leptin-melanocortin pathway is pivotal in appetite and energy homeostasis. Pathogenic variants in genes involved in this pathway lead to severe early-onset monogenic obesity (MO). The MC4R gene plays a central role in leptin-melanocortin signaling, and heterozygous variants in this gene are the most common cause of MO. A targeted gene panel consisting of 52 obesity-related genes was used to screen for variants associated with obesity. Variants were analyzed and filtered to identify potential disease-causing activity and validated using Sanger sequencing. We identified two novel heterozygous variants, c.253A>G p.Ser85Gly and c.802T>C p.Tyr268His, in the MC4R gene in two unrelated patients with morbid obesity and evaluated the functional impact of these variants. The impact of the variants on the MC4R gene was assessed using in silico prediction tools and molecular dynamics simulation. To further study the pathogenicity of the identified variants, GT1-7 cells were transfected with plasmid DNA encoding either wild-type or mutant MC4R variants. The effects of allelic variations in the MC4R gene on cAMP synthesis, MC4R protein level, and activation of PKA, ERB, and CREB signaling pathways in both stimulated and unstimulated ɑ-MSH paradigms were determined for their functional implications. In silico analysis suggested that the variants destabilized the MC4R structure and affected the overall dynamics of the MC4R protein, possibly leading to intracellular receptor retention. In vitro analysis of the functional impact of these variants showed a significant reduction in cell surface receptor expression and impaired extracellular ligand binding activity, leading to reduced cAMP production. Our analysis shows that the variants do not affect total protein expression; however, they are predicted to affect the post-translational localization of the MC4R protein to the cell surface and impair downstream signaling cascades such as PKA, ERK, and CREB signaling pathways. This finding might help our patients to benefit from the novel therapeutic advances for monogenic forms of obesity.

A nonequilibrium allosteric model for receptor-kinase complexes: The role of energy dissipation in chemotaxis signaling

The Escherichia coli chemotaxis signaling pathway has served as a model system for the adaptive sensing of environmental signals by large protein complexes. The chemoreceptors control the kinase activity of CheA in response to the extracellular ligand concentration and adapt across a wide concentration range by undergoing methylation and demethylation. Methylation shifts the kinase response curve by orders of magnitude in ligand concentration while incurring a much smaller change in the ligand binding curve. Here, we show that the disproportionate shift in binding and kinase response is inconsistent with equilibrium allosteric models. To resolve this inconsistency, we present a nonequilibrium allosteric model that explicitly includes the dissipative reaction cycles driven by adenosine triphosphate (ATP) hydrolysis. The model successfully explains all existing joint measurements of ligand binding, receptor conformation, and kinase activity for both aspartate and serine receptors. Our results suggest that the receptor complex acts as an enzyme: Receptor methylation modulates the ON-state kinetics of the kinase (e.g., phosphorylation rate), while ligand binding controls the equilibrium balance between kinase ON/OFF states. Furthermore, sufficient energy dissipation is responsible for maintaining and enhancing the sensitivity range and amplitude of the kinase response. We demonstrate that the nonequilibrium allosteric model is broadly applicable to other sensor-kinase systems by successfully fitting previously unexplained data from the DosP bacterial oxygen-sensing system. Overall, this work provides a nonequilibrium physics perspective on cooperative sensing by large protein complexes and opens up research directions for understanding their microscopic mechanisms through simultaneous measurements and modeling of ligand binding and downstream responses.

Hallucination of closed repeat proteins containing central pockets.

In pseudocyclic proteins, such as TIM barrels, β barrels, and some helical transmembrane channels, a single subunit is repeated in a cyclic pattern, giving rise to a central cavity that can serve as a pocket for ligand binding or enzymatic activity. Inspired by these proteins, we devised a deep-learning-based approach to broadly exploring the space of closed repeat proteins starting from only a specification of the repeat number and length. Biophysical data for 38 structurally diverse pseudocyclic designs produced in Escherichia coli are consistent with the design models, and the three crystal structures we were able to obtain are very close to the designed structures. Docking studies suggest the diversity of folds and central pockets provide effective starting points for designing small-molecule binders and enzymes.