Fluorescent Peptide Research Articles

Pyrene-Modified Cyclic Peptides Detect Cu2+ Ions by Fluorescence in Water

The detection of metal ions is an option for maintaining water quality and diagnosing metal ion-related diseases. In this study, we successfully detected metal ions using fluorescent peptides in water. First, we prepared seven linear (L1–L7) and seven cyclic (C1–C7) peptides containing two pyrenyl (Pyr) units and assessed the response to various metal ions by fluorescence. The results indicated that C1, which contains a hexameric cyclic peptide moiety consisting of Pyr and Gly units, did not show a fluorescent response to metal ions, while the linear L1 corresponding to C1 showed a response to Cu2+, but its selectivity was found to be poor through a competition assay for each metal ion. We then assessed C2–C7 and L2–L7, in which Gly was replaced by His units at various positions in the same manner. The results showed that C2–C7 responded to Cu2+ in a manner dependent on the His position. Additionally, superior selectivity was observed in C7 through a competition assay. These results demonstrate that the structural restriction of peptides and the sequence affect the selective detection of Cu2+ and reveal that peptides with an appropriate structure can accomplish the fluorescent detection of Cu2+ specifically.

New Salicylanilide Derivatives and Their Peptide Conjugates as Anticancer Compounds: Synthesis, Characterization, and In Vitro Effect on Glioblastoma.

Pharmacologically active salicylanilides (2-hydroxy-N-phenylbenzamides) have been a promising area of interest in medicinal chemistry-related research for quite some time. This group of compounds has shown a wide spectrum of biological activities, including but not limited to anticancer effects. In this study, substituted salicylanilides were chosen to evaluate the in vitro activity on U87 human glioblastoma (GBM) cells. The parent salicylanilide, salicylanilide 5-chloropyrazinoates, a 4-aminosalicylic acid derivative, and the new salicylanilide 4-formylbenzoates were chemically and in vitro characterized. To enhance the internalization of the compounds, they were conjugated to delivery peptides with the formation of oxime bonds. Oligotuftsins ([TKPKG]n, n = 1-4), the ligands of neuropilin receptors, were used as GBM-targeting carrier peptides. The in vitro cellular uptake, intracellular localization, and penetration ability on tissue-mimicking models of the fluorescent peptide derivatives were determined. The compounds and their peptide conjugates significantly decreased the viability of U87 glioma cells. Salicylanilide compound-induced GBM cell death was associated with activation of autophagy, as characterized by immunodetection of autophagy-related processing of light chain 3 protein.

Exploring the therapeutic potential of DV-B-120 as an inhibitor of dengue virus infection.

Dengue fever, an infectious disease prevalent in subtropical and tropical regions, currently lacks effective small-molecule drugs as treatment. In this study, we used a fluorescence peptide cleavage assay to screen seven compounds to assess their inhibition of the dengue virus (DENV) NS2B-NS3 protease. DV-B-120 demonstrated superior inhibition of NS2B-NS3 protease activity and lower toxicity compared to ARDP0006. The selectivity index of DV-B-120 was higher than that of ARDP0006. In vivo assessments of the antiviral efficacy of DV-B-120 against DENV replication demonstrated delayed mortality of suckling mice treated with the compound, with 60-80% protection against life-threatening effects, compared to the outcomes of DENV-infected mice treated with saline. The lower clinical scores of DENV-infected mice treated with DV-B-120 indicated a reduction in acute-progressive illness symptoms, underscoring the potential therapeutic impact of DV-B-120. Investigations of DV-B-120's ability to restore the antiviral type I IFN response in the brain tissue of DENV-infected ICR suckling mice demonstrated its capacity to stimulate IFN and antiviral IFN-stimulated gene expression. DV-B-120 not only significantly delayed DENV-2-induced mortality and illness symptoms but also reduced viral numbers in the brain, ultimately restoring the innate antiviral response. These findings strongly suggest that DV-B-120 holds promise as a therapeutic agent against DENV infection and highlight its potential contribution in addressing the current lack of effective treatments for this infectious disease.IMPORTANCEThe prevalence of dengue virus (DENV) infection in tropical and subtropical regions is escalating due to factors like climate change and mosquito vector expansion. With over 300 million annual infections and potentially fatal outcomes, the urgent need for effective treatments is evident. While the approved Dengvaxia vaccine has variable efficacy, there are currently no antiviral drugs for DENV. This study explores seven compounds targeting the NS2B-NS3 protease, a crucial protein in DENV replication. These compounds exhibit inhibitory effects on DENV-2 NS2B-NS3, holding promise for disrupting viral replication and preventing severe manifestations. However, further research, including animal testing, is imperative to assess therapeutic efficacy and potential toxicity. Developing safe and potent treatments for DENV infection is critical in addressing the rising global health threat posed by this virus.

Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y4 receptor (Y4R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y4R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y4R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y4R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y4R ligands derived from recently reported cyclic hexapeptides showing picomolar Y4R binding affinity. With pKi values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y4R affinity. Y4R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y4R (equilibrium pKd: 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y4R competition binding studies and imaging techniques such as single-receptor molecule tracking.

Steady State and Time-Dependent Fluorescent Peptide Assays for Protein Kinases.

Protein kinases catalyze the phosphorylation of proteins most commonly on Ser, Thr, and Tyr residues and regulate many cellular events in eukaryotic cells, such as cell cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP-binding site and one or more substrate-binding site(s) that exhibit recognition features for different protein substrates. By bringing ATP and a substrate into proximity, each protein kinase can transfer the γ phosphate of the ATP molecule to a hydroxyl group of the target residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated based on the phosphorylated versus dephosphorylated status of the substrate. Although there are a number of ways to assay the activity of protein kinases, most of them are technically cumbersome and/or are indirect or based on quenched reactions. This protocol describes an assay employing a fluorescent peptide substrate to detect phosphorylation by protein kinases in real time. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore. We extend the application of this assay to an example of how to assess time-dependent covalent inhibition of kinases as well. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Measuring protein kinase activity using fluorescent peptides Alternate Protocol: Measuring protein kinase activity using a fluorescence plate reader Support Protocol: Labeling peptides with sox fluorophore Basic Protocol 2: Measuring time-dependent ATP-competitive inhibition of protein kinases using fluorescent peptides.

Design, Synthesis, and Anticancer Activity of N‐1 Terminal Segment of α‐(2, 5,7‐Tri‐tert‐butylindol‐3‐yl)alanine‐Incorporated Short Peptides

AbstractUsing a minimallised approach,we designed and synthesized N‐terminal modified peptides W‐KRGNKWLA‐CONH2 (P1) and W‐ARGDGGNGRGA‐CONH2 (P2) using Fmoc‐Chemistry solid phase peptide synthesis protocol. High‐performance liquid chromatograms and mass spectrometry (LCMS and analytical HPLC) were employed to determine the purity and molecular weight of the peptide. Surface tension measurements are used to evaluate peptide surface activity. The primary structure of the peptideis anlaysed by NMR spectroscopy. Circular dichroism studies demonstrate that the secondary structure of the peptide changes minimally when it attached to anionic lipid bilayers (POPC : POPG). According to a steady‐state fluorescence experiment, peptides P1 and P2 preferentially interact with anionic (POPC : POPG) lipid bilayers rather than zwitterionic (POPC) lipid bilayers. The peptide P2 interacts preferentially with anionic lipid membranes and has an anticancer effect due to RGD and NGR binding motifs. The cytotoxicity of the MTT experiment demonstrated that both normal and cancerous cells could lower peptide concentrations below their IC50 levels. Molecular docking also supports in‐vitro anticancer research. Both peptides P1 and P2 were non‐toxic to vero cells and appeared to be more promising as it demonstrated wide spectrum anticancer activity.

A Modified Tridecapeptide Probe for Imaging Cell Junction.

Cell junctions, which are typically associated with dynamic cytoskeletons, are essential for a wide range of cellular activities, including cell migration, cell communication, barrier function and signal transduction. Observing cell junctions in real-time can help us understand the mechanisms by which they regulate these cellular activities. This study examined the binding capacity of a modified tridecapeptide from Connexin 43 (Cx43) to the cell junction protein zonula occludens-1 (ZO-1). The goal was to create a fluorescent peptide that can label cell junctions. A cell-penetrating peptide was linked to the modified tridecapeptide. The heterotrimeric peptide molecule was then synthesized. The binding of the modified tridecapeptide was tested using pulldown and immunoprecipitation assays. The ability of the peptide to label cell junctions was assessed by adding it to fixed or live Caco-2 cells. The testing assays revealed that the Cx43-derived peptide can bind to ZO-1. Additionally, the peptide was able to label cell junctions of fixed cells, although no obvious cell junction labeling was observed clearly in live cells, probably due to the inadequate affinity. These findings suggest that labeling cell junctions using a peptide-based strategy is feasible. Further efforts to improve its affinity are warranted in the future.

Chemo-enzymatic synthesis of tetrasaccharide linker peptides to study the divergent step in glycosaminoglycan biosynthesis.

Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.

Hidden imaging in thin polymer films with embedded fluorescent peptide nanodots.

Fluorescent (FL) encrypting nanostructures, such as quantum dots, carbon dots, organic dyes, lanthanide nanocrystals, DNA, and more, are effective tools for advanced applications in high-resolution hidden imaging. These applications include tracking, labeling, security printing, and anti-counterfeiting drug technology. In this work, what we believe to be a new FL encoding nanostructures has been proposed, which consists of recently discovered nanometer-scale peptide dots. When refolded into a beta-sheet peptide secondary structure, these biocompatible nanoparticles exhibit a strong and tunable FL effect. The biophotonic FL covers the entire visible spectrum, making the peptide dots next-generation nanoscale light sources with a quantum yield of 30%. Our studies demonstrate that these FL bio-nanodots also exhibit a significant irreversible photo-bleaching effect associated with the light-induced destruction of noncovalent intermolecular hydrogen bonds of the peptide dots' highly stable beta-sheet secondary structure. We present what we believe is a new approach for achieving high-resolution long-term optical memory by tailoring various hidden images in the developed thin polyvinyl alcohol (PVA) polymer films with an embedded dense array of FL peptide nanodots. The technology enables recording photo-bleached patterns, barcodes, and high-resolution images.

Development of an environmentally sensitive fluorescent peptide probe for MrgX2 and application in ligand screening of peptide antibiotics

Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.

Molecular skin fluorescence imaging: A tool for evaluating early melanoma development.

A novel approach to melanoma diagnosis-in vivo molecular skin fluorescence imaging (mSFI)-was developed to identify premalignant changes in the form of tissue remodeling related to melanoma development in humans by imaging the proximal microenvironment of lesions. The method was tested using a fluorescent peptide (ORL-1) which binds to αvβ3 integrin, a molecule associated with invasive melanoma development. A cut off score of 7 was established, differentiating melanomas from nonmelanoma nevi with 100% sensitivity, and 95.7% specificity, while identifying dysplastic nevi with the potential for melanoma development.

Ligand reaction-based fluorescent peptide probes for the detection of Cu2+ and glutathione.

Copper is a critical element in both human and animal metabolic processes. Its role includes supporting connective tissue cross-linking, as well as iron and lipid metabolism; at the same time, copper is also a toxic heavy metal that can cause harm to both the environment and human health. Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine, and glycine combined with sulfhydryl groups. Its properties include acting as an antioxidant and facilitating integrative detoxification. GSH is present in both plant and animal cells and has a fundamental role in maintaining living organisms. GSH is the most abundant thiol antioxidant in the human body. It exists in reduced and oxidized forms within cells and provides significant biochemical functions, such as regulating vitamins such as vitamins D, E, and C, and facilitating detoxification. A fluorescent probe has been developed to detect copper ions selectively, sensitively, and rapidly. This report outlines the successful work on creating a peptide probe, TGN (TPE-Trp-Pro-Gly-Cln-His-NH2 ), with specific Cu2+ detection capabilities, and a significant fluorescence recovery occurred with the addition of GSH. This indicates that the probe can detect Cu2+ and GSH concurrently. The detection limit for Cu2+ in the buffer solution was 264 nM (R2 = 0.9992), and the detection limit for GSH using the TGN-Cu2+ complex was 919 nM (R2 = 0.9917). The probe exhibits high cell permeability and low biotoxicity that make it ideal for live cell imaging in biological conditions. This peptide probe has the capability to detect Cu2+ and GSH in biological cells.

A novel “off–on” peptide fluorescent probe for the detection of copper and sulphur ions in living cells

The fluorescent peptide probe FGP1 quickly detects Cu2+ ions, leading to visible fluorescence quenching. The addition of S2− ions to the FGP1-Cu complex restores fluorescence, resulting in an 'off–on' process.

Fluorescent peptide for detecting factor XIIIa activity and fibrin in whole blood clots forming under flow

BackgroundDuring clotting, thrombin generates fibrin monomers and activates plasma-derived transglutaminase factor (F) XIIIa; collagen and thrombin-activated platelets offer thrombin-independent cellular FXIIIa (cFXIIIa) for clotting. Detecting fibrin on collagen and tissue factor surfaces in whole blood clotting typically uses complex reagents like fluorescent fibrinogen or antifibrin antibody. ObjectivesWe want to test whether the peptide using the α2- antiplasmin crosslinking mechanism by FXIIIa is a useful tool in both monitoring FXIIIa activity, and visualize and monitor fibrin formation, deposition, and extent of crosslinking within fibrin structures in whole blood clots formed under flow. MethodsWe tested a fluorescent peptide derived from α2-antiplasmin sequence (Ac-GNQEQVSPLTLLKWC-fluorescein) to monitor the location of transglutaminase activity and fibrin during whole blood clotting under microfluidic flow (wall shear rate, 100 s−1). ResultsThe peptide rapidly colocated with accumulating fibrin due to transglutaminase activity, confirmed by Phe-Pro-Arg-chloromethylketone inhibiting fibrin and peptide labeling. The FXIIIa inhibitor T101 had no effect on fibrin generation but ablated the labeling of fibrin by the peptide. Similarly, Gly-Pro-Arg-Pro abated fibrin formation and thus strongly attenuated the peptide signal. At arterial wall shear rate (1000 s−1), less fibrin was formed, and consequently, less peptide labeling of fibrin was detected compared with venous conditions. The addition of tissue plasminogen activator caused a reduction of both fibrin and peptide signals. Also, the peptide strongly colocalized with fibrin (but not platelets) in clots from laser-injured mouse cremaster arterioles. For clotting under flow, FXIIIa activity was most likely plasma-derived since a RhoA inhibitor did not block α2-antiplasmin fragment cross-linking to fibrin. ConclusionUnder flow, the majority of FXIIIa-dependent fibrin labeling with peptide during clotting was distal of thrombin activity. The synthetic peptide provided a strong and sustained labeling of fibrin as it formed under flow.

Norbornene-chitosan spray-dried microspheres for peptide conjugation using thiol-ene “photoclick” chemistry

The action of bioactive peptides, such as antimicrobial peptides (AMP), in the human body is often compromised by limited residence time and stability in the target site. Bioconjugation of peptides to biomaterial surfaces is one of the strategies that may overcome these limitations. Herein, norbornene-chitosan (NorChit) microspheres were engineered to react with thiolated peptides by thiol-ene “photoclick” chemistry. NorChit microspheres were produced by spray drying and crosslinked with dithiothreitol (DTT) to prevent their solubilization. Microspheres with a diameter of 5 ± 2 µm showed round and smooth morphology with pockets over the surface that could be related with hydrophobic interactions between internal norbornene groups. Thiol-ene bioconjugation carried out using a fluorescent model peptide, showed a yield of 45%, whereas using the peptide but without UV exposure indicated a maximum of peptide adsorption of 30%. Altogether, NorChit microspheres show the potential for carrying bioactive peptides, which may open avenues for AMP activity onto harsh environments in the body. The action of bioactive peptides, such as antimicrobial peptides (AMP), in the human body is often compromised by limited residence time and stability in the target site. Bioconjugation of peptides to biomaterial surfaces is one of the strategies that may overcome these limitations. Herein, norbornene-chitosan (NorChit) microspheres were engineered to react with thiolated peptides by thiol-ene “photoclick” chemistry. NorChit microspheres were produced by spray drying and crosslinked with dithiothreitol (DTT) to prevent their solubilization. Microspheres with a diameter of 5 ± 2 μm showed round and smooth morphology with pockets over the surface that could be related with hydrophobic interactions between internal norbornene groups. Thiol-ene bioconjugation carried out using a fluorescent model peptide, showed a yield of 45%, whereas using the peptide but without UV exposure indicated a maximum of peptide adsorption of 30%. Altogether, NorChit microspheres show the potential for carrying bioactive peptides, which may open avenues for AMP activity onto harsh environments in the body.

Synthesis of Fluorescent Cyclic Peptides via Gold(I)-Catalyzed Macrocyclization.

Rapid and efficient cyclization methods that form structurally novel peptidic macrocycles are of high importance for medicinal chemistry. Herein, we report the first gold(I)-catalyzed macrocyclization of peptide-EBXs (ethynylbenziodoxolones) via C2-Trp C-H activation. This reaction was carried out in the presence of protecting group free peptide sequences and is enabled by a simple commercial gold catalyst (AuCl·Me2S). The method displayed a rapid reaction rate (within 10 min), wide functional group tolerance (27 unprotected peptides were cyclized), and up to 86% isolated yield. The obtained highly conjugated cyclic peptide linker, formed through C-H alkynylation, can be directly applied to live-cell imaging as a fluorescent probe without further attachment of fluorophores.

High-Throughput Identification of Graft-Vs-Leukemia TCRs in AML Patient Samples after Allogeneic Stem Cell Transplantation

For patients with intermediate and high-risk AML/MDS, allogeneic stem cell transplantation (aSCT) is a potentially curative therapy. Efficacy is driven by the graft-versus-leukemia (GvL) response, in which donor-derived T cells recognize peptide:MHC antigens presented by leukemia cells, and subsequently kill them. The antigens that drive GvL responses are attractive therapeutic targets for engineered cell therapies and/or biologics. However, only a small number of GvL antigens have been identified, limiting the therapeutic potential of targeted immunotherapies. Deep interrogation of the T cell repertoires of patients who have responded favorably to aSCT presents an opportunity to identify bona fide GvL antigens, but high-throughput methods need to be developed to do this effectively. A popular method to identify antigen-specific T cells is fluorescent peptide:MHC tetramer staining, whereby T cells whose T cell receptors (TCR) bind a particular peptide:MHC antigen become fluorescent and identifiable by flow cytometry. However, a major challenge to identifying GvL T cells using tetramers is that the prediction of immunogenic antigens - that is, those that induce a T cell response in vivo - is poor; studies of solid tumors suggest that only ~1% of predicted MHC-binding neoantigens are immunogenic in patients. In AML/MDS aSCT patients, there are thousands of peptides that are predicted to bind MHCI and represent potential GvL antigens, but due to practical limitations of patient blood and bone marrow sample quantities, only a small subset can be screened by flow cytometry. Other higher throughput methods using heavy-metal tagged peptide:MHC tetramers followed by mass cytometry, or DNA-barcoded tetramers followed by bulk sequencing have been employed to increase antigen screening scale, but these methods are limited in sensitivity and destroy the T cell without providing TCR sequences. To overcome these limitations and rapidly identify GvL T cells in patient samples, we developed a single cell sequencing based method that enables the screening of thousands of peptide:MHC antigens simultaneously, using a single patient sample. We generated peptide: MHCI monomers using UV-mediated peptide exchange and generated large panels of combinatorial DNA-barcoded peptide:MHCI tetramers at microscale. We integrated our methodology into the 10X Genomics Single Cell Immune Profiling workflow to enable simultaneous recovery of TCR antigen-specificity, paired TCR alpha beta sequences, and single cell RNA-Seq data. These methods were used to screen post-transplant AML patient blood and bone marrow, and we identified expanded GvL T cell clonotypes that target novel minor histocompatibility antigens, overexpressed “aberrant self” proteins, and other leukemia associated antigens. We generated primary CD8 T cells that retrovirally express these TCRs and demonstrate that they are potent and have the expected antigen specificities. The methods described are highly sensitive, specific, efficient, and scalable, and enable high confidence identification of antigen specific clonotypes in patient samples. They markedly expand the throughput compared to existing methods, and simultaneously yield paired alpha beta TCR sequences, facilitating a full characterization of the antigen-TCR drivers of GvL. Furthermore, these methods have broad utility beyond SCT, as they are applicable to studying T cell responses to other immune settings, including anti-cancer, autoimmune, and anti-microbial responses.

In Situ Generation of Fluorescent Amino Acids and Peptides via Double C–H Activation/Annulation

In Situ Generation of Fluorescent Amino Acids and Peptides via Double C–H Activation/Annulation

Synthesis, Activity, and Application of Fluorescent Analogs of [D1G, Δ14Q]LvIC Targeting α6β4 Nicotinic Acetylcholine Receptor.

α6β4* nicotinic acetylcholine receptor (nAChR) (* represents the possible presence of additional subunits) is mainly distributed in the central and peripheral nervous system and is associated with neurological diseases, such as neuropathic pain; however, the ability to explore its function and distribution is limited due to the lack of pharmacological tools. As one of the analogs of α-conotoxin (α-CTx) LvIC from Conus lividus, [D1G, Δ14Q]LvIC (Lv) selectively and potently blocks α6/α3β4 nAChR (α6/α3 represents a chimera). Here, we synthesized three fluorescent analogs of Lv by connecting fluorescent molecules 6-carboxytetramethylrhodamine succinimidyl ester (6-TAMRA-SE, R), Cy3 NHS ester (Cy3, C) and BODIPY-FL NHS ester (BDP, B) to the N-terminus of the peptide and obtained Lv-R, Lv-C, and Lv-B, respectively. The potency and selectivity of three fluorescent peptides were evaluated using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes, and the potency and selectivity of Lv-B were almost maintained with the half-maximal inhibition (IC50) of 64 nM. Then, we explored the stability of Lv-B in artificial cerebrospinal fluid and stained rat brain slices with Lv-B. The results indicated that the stability of Lv-B was slightly improved compared to that of native Lv. Additionally, we detected the distribution of the α6β4* nAChR subtype in the cerebral cortex using green fluorescently labeled peptide and fluorescence microscopy. Our findings not only provide a visualized pharmacological tool for exploring the distribution of the α6β4* nAChR subtype in various situ tissues and organs but also extend the application of α-CTx [D1G, Δ14Q]LvIC to demonstrate the involvement of α6β4 nAChR function in pathophysiology and pharmacology.

Cellular Uptake of a Fluorescent Ligand Reveals Ghrelin O-Acyltransferase Interacts with Extracellular Peptides and Exhibits Unexpected Localization for a Secretory Pathway Enzyme.

Ghrelin O-acyltransferase (GOAT) plays a central role in the maturation and activation of the peptide hormone ghrelin, which performs a wide range of endocrinological signaling roles. Using a tight-binding fluorescent ghrelin-derived peptide designed for high selectivity for GOAT over the ghrelin receptor GHSR, we demonstrate that GOAT interacts with extracellular ghrelin and facilitates ligand cell internalization in both transfected cells and prostate cancer cells endogenously expressing GOAT. Coupled with enzyme mutagenesis, ligand uptake studies support the interaction of the putative histidine general base within GOAT with the ghrelin peptide acylation site. Our work provides a new understanding of GOAT's catalytic mechanism, establishes that GOAT can interact with ghrelin and other peptides located outside the cell, and raises the possibility that other peptide hormones may exhibit similar complexity in their intercellular and organismal-level signaling pathways.