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Nature Communications Apr 2020Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody's specificity. Here we develop a new...
Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody's specificity. Here we develop a new type of conjugate that consists of a nanobody and a peptidic ligand for a G protein-coupled receptor (GPCR), fused via their C-termini. We address activation of parathyroid hormone receptor-1 (PTHR1) and improve the signaling activity and specificity of otherwise poorly active N-terminal peptide fragments of PTH by conjugating them to nanobodies (VHHs) that recognize PTHR1. These C-to-C conjugates show biological activity superior to that of the parent fragment peptide in vitro. In an exploratory experiment in mice, a VHH-PTH peptide conjugate showed biological activity, whereas the corresponding free peptide did not. The lead conjugate also possesses selectivity for PTHR1 superior to that of PTH(1-34). This design approach, dubbed "conjugation of ligands and antibodies for membrane proteins" (CLAMP), can yield ligands with high potency and specificity.
Topics: Amino Acid Sequence; Animals; Female; HEK293 Cells; Humans; Ligands; Mice; Models, Molecular; Parathyroid Hormone; Peptides; Protein Binding; Receptor, Parathyroid Hormone, Type 1; Receptors, G-Protein-Coupled; Single-Domain Antibodies
PubMed: 32350260
DOI: 10.1038/s41467-020-15884-8 -
Organic & Biomolecular Chemistry Jan 2021DNAzymes were previously identified by in vitro selection for a variety of chemical reactions, including several biologically relevant peptide modifications. However,...
DNAzymes were previously identified by in vitro selection for a variety of chemical reactions, including several biologically relevant peptide modifications. However, finding DNAzymes for peptide lysine acylation is a substantial challenge. By using suitably reactive aryl ester acyl donors as the electrophiles, here we used in vitro selection to identify DNAzymes that acylate amines, including lysine side chains of DNA-anchored peptides. Some of the DNAzymes can transfer a small glutaryl group to an amino group. These results expand the scope of DNAzyme catalysis and suggest the future broader applicability of DNAzymes for sequence-selective lysine acylation of peptide and protein substrates.
Topics: Acylation; Amines; Biocatalysis; DNA, Catalytic; Lysine; Peptides
PubMed: 33150349
DOI: 10.1039/d0ob02015j -
Journal of Chemical Theory and... Jun 2022An emerging class of therapeutic molecules are cyclic peptides with over 40 cyclic peptide drugs currently in clinical use. Their mode of action is, however, not fully...
An emerging class of therapeutic molecules are cyclic peptides with over 40 cyclic peptide drugs currently in clinical use. Their mode of action is, however, not fully understood, impeding rational drug design. Computational techniques could positively impact their design, but modeling them and their interactions remains challenging due to their cyclic nature and their flexibility. This study presents a step-by-step protocol for generating cyclic peptide conformations and docking them to their protein target using HADDOCK2.4. A dataset of 30 cyclic peptide-protein complexes was used to optimize both cyclization and docking protocols. It supports peptides cyclized via an N- and C-terminus peptide bond and/or a disulfide bond. An ensemble of cyclic peptide conformations is then used in HADDOCK to dock them onto their target protein using knowledge of the binding site on the protein side to drive the modeling. The presented protocol predicts at least one acceptable model according to the critical assessment of prediction of interaction criteria for each complex of the dataset when the top 10 HADDOCK-ranked single structures are considered (100% success rate top 10) both in the bound and unbound docking scenarios. Moreover, its performance in both bound and fully unbound docking is similar to the state-of-the-art software in the field, Autodock CrankPep. The presented cyclization and docking protocol should make HADDOCK a valuable tool for rational cyclic peptide-based drug design and high-throughput screening.
Topics: Cyclization; Molecular Docking Simulation; Peptides, Cyclic; Protein Binding; Protein Conformation; Proteins; Software
PubMed: 35652781
DOI: 10.1021/acs.jctc.2c00075 -
Methods in Enzymology 2022A protein's structure and function often depend not only on its primary sequence, but also the presence or absence of any number of non-coded posttranslational...
A protein's structure and function often depend not only on its primary sequence, but also the presence or absence of any number of non-coded posttranslational modifications. Complicating their study is the fact that the physiological consequences of these modifications are context-, protein-, and site-dependent, and there exist no purely biological techniques to unambiguously study their effects. To this end, protein semisynthesis has become an invaluable chemical biology tool to specifically install non-coded or non-native moieties onto proteins in vitro using synthetic and/or recombinant polypeptides. Here, we describe two facets of protein semisynthesis (solid-phase peptide synthesis and expressed protein ligation) and their use in generating site-specifically glycosylated small heat shock proteins for functional studies. The procedures herein require limited specialized equipment, employ mild reaction conditions, and can be extended to myriad other proteins, modifications, and contexts.
Topics: Heat-Shock Proteins, Small; Peptides; Protein Processing, Post-Translational; Proteins
PubMed: 36220281
DOI: 10.1016/bs.mie.2022.07.004 -
Biochemical Society Transactions Oct 2020The conformation with which natural agonistic peptides interact with G protein-coupled receptor(s) (GPCR(s)) partly results from intramolecular interactions such as... (Review)
Review
The conformation with which natural agonistic peptides interact with G protein-coupled receptor(s) (GPCR(s)) partly results from intramolecular interactions such as hydrogen bridges or is induced by ligand-receptor interactions. The conformational freedom of a peptide can be constrained by intramolecular cross-links. Conformational constraints enhance the receptor specificity, may lead to biased activity and confer proteolytic resistance to peptidic GPCR agonists. Chemical synthesis allows to introduce a variety of cross-links into a peptide and is suitable for bulk production of relatively simple lead peptides. Lanthionines are thioether bridged alanines of which the two alanines can be introduced at different distances in chosen positions in a peptide. Thioether bridges are much more stable than disulfide bridges. Biosynthesis of lanthionine-constrained peptides exploiting engineered Gram-positive or Gram-negative bacteria that contain lanthionine-introducing enzymes constitutes a convenient method for discovery of lanthionine-stabilized GPCR agonists. The presence of an N-terminal leader peptide enables dehydratases to dehydrate serines and threonines in the peptide of interest after which a cyclase can couple the formed dehydroamino acids to cysteines forming (methyl)lanthionines. The leader peptide also guides the export of the formed lanthionine-containing precursor peptide out of Gram-positive bacteria via a lanthipeptide transporter. An engineered cleavage site in the C-terminus of the leader peptide allows to cleave off the leader peptide yielding the modified peptide of interest. Lanthipeptide GPCR agonists are an emerging class of therapeutics of which a few examples have demonstrated high efficacy in animal models of a variety of diseases. One lanthipeptide GPCR agonist has successfully passed clinical Phase Ia.
Topics: Alanine; Animals; Anti-Bacterial Agents; Bacterial Proteins; Disulfides; Drug Discovery; GTP-Binding Proteins; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Lanthanoid Series Elements; Membrane Transport Proteins; Mice; Peptide Hydrolases; Peptides; Protein Domains; Protein Processing, Post-Translational; Protein Sorting Signals; Rats; Receptors, G-Protein-Coupled; Substrate Specificity; Sulfides
PubMed: 33125486
DOI: 10.1042/BST20200427 -
Journal of the American Chemical Society Apr 2021Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like, threaded conformation....
Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like, threaded conformation. Owing to a locked three-dimensional structure, lasso peptides can be unusually stable toward heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. All known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent class of RiPPs; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address longstanding challenges associated with lasso peptide production. We report the successful use of CFB for the formation of an array of sequence-diverse lasso peptides that include known examples as well as a new predicted lasso peptide from . We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants to evaluate the substrate tolerance of the biosynthetic pathway. By evaluating more than 1000 randomly chosen variants, we show that the lasso-forming cyclase from the fusilassin pathway is capable of producing millions of sequence-diverse lasso peptides via CFB. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.
Topics: Amino Acid Sequence; Bacterial Proteins; Biological Products; Cell-Free System; Cyclization; Multigene Family; Peptide Hydrolases; Peptides; Protein Processing, Post-Translational; Ribosomes; Substrate Specificity; Thermobifida
PubMed: 33823110
DOI: 10.1021/jacs.1c01452 -
Cellular and Molecular Life Sciences :... Aug 2022Ubiquitin is a small, globular protein that is conjugated to other proteins as a posttranslational event. A palette of small, folded domains recognizes and binds...
Ubiquitin is a small, globular protein that is conjugated to other proteins as a posttranslational event. A palette of small, folded domains recognizes and binds ubiquitin to translate and effectuate this posttranslational signal. Recent computational studies have suggested that protein regions can recognize ubiquitin via a process of folding upon binding. Using peptide binding arrays, bioinformatics, and NMR spectroscopy, we have uncovered a disordered ubiquitin-binding motif that likely remains disordered when bound and thus expands the palette of ubiquitin-binding proteins. We term this motif Disordered Ubiquitin-Binding Motif (DisUBM) and find it to be present in many proteins with known or predicted functions in degradation and transcription. We decompose the determinants of the motif showing it to rely on features of aromatic and negatively charged residues, and less so on distinct sequence positions in line with its disordered nature. We show that the affinity of the motif is low and moldable by the surrounding disordered chain, allowing for an enhanced interaction surface with ubiquitin, whereby the affinity increases ~ tenfold. Further affinity optimization using peptide arrays pushed the affinity into the low micromolar range, but compromised context dependence. Finally, we find that DisUBMs can emerge from unbiased screening of randomized peptide libraries, featuring in de novo cyclic peptides selected to bind ubiquitin chains. We suggest that naturally occurring DisUBMs can recognize ubiquitin as a posttranslational signal to act as affinity enhancers in IDPs that bind to folded and ubiquitylated binding partners.
Topics: Amino Acid Sequence; Intrinsically Disordered Proteins; Peptides; Protein Binding; Proteins; Ubiquitin
PubMed: 35974206
DOI: 10.1007/s00018-022-04486-w -
International Journal of Molecular... Mar 2021Obesity/overweight are important health problems due to metabolic complications. Dysregulation of peptides exerting orexigenic/anorexigenic effects must be investigated... (Review)
Review
Obesity/overweight are important health problems due to metabolic complications. Dysregulation of peptides exerting orexigenic/anorexigenic effects must be investigated in-depth to understand the mechanisms involved in feeding behaviour. One of the most important and studied orexigenic peptides is galanin (GAL). The aim of this review is to update the mechanisms of action and physiological roles played by the GAL family of peptides (GAL, GAL-like peptide, GAL message-associated peptide, alarin) in the control of food intake and to review the involvement of these peptides in metabolic diseases and food intake disorders in experimental animal models and humans. The interaction between GAL and NPY in feeding and energy metabolism, the relationships between GAL and other substances involved in food intake mechanisms, the potential pharmacological strategies to treat food intake disorders and obesity and the possible clinical applications will be mentioned and discussed. Some research lines are suggested to be developed in the future, such as studies focused on GAL receptor/neuropeptide Y Y receptor interactions in hypothalamic and extra-hypothalamic nuclei and sexual differences regarding the expression of GAL in feeding behaviour. It is also important to study the possible GAL resistance in obese individuals to better understand the molecular mechanisms by which GAL regulates insulin/glucose metabolism. GAL does not exert a pivotal role in weight regulation and food intake, but this role is crucial in fat intake and also exerts an important action by regulating the activity of other key compounds under conditions of stress/altered diet.
Topics: Animals; Eating; Energy Metabolism; Galanin; Humans; Hypothalamus; Neuropeptide Y; Obesity
PubMed: 33802616
DOI: 10.3390/ijms22052544 -
Molecules (Basel, Switzerland) May 2020The high affinity and specificity of peptides towards biological targets, in addition to their favorable pharmacological properties, has encouraged the development of... (Review)
Review
The high affinity and specificity of peptides towards biological targets, in addition to their favorable pharmacological properties, has encouraged the development of many peptide-based pharmaceuticals, including peptide-based positron emission tomography (PET) radiopharmaceuticals. However, the poor in vivo stability of unmodified peptides against proteolysis is a major challenge that must be overcome, as it can result in an impractically short in vivo biological half-life and a subsequently poor bioavailability when used in imaging and therapeutic applications. Consequently, many biologically and pharmacologically interesting peptide-based drugs may never see application. A potential way to overcome this is using peptide analogues designed to mimic the pharmacophore of a native peptide while also containing unnatural modifications that act to maintain or improve the pharmacological properties. This review explores strategies that have been developed to increase the metabolic stability of peptide-based pharmaceuticals. It includes modifications of the - and/or -termini, introduction of d- or other unnatural amino acids, backbone modification, PEGylation and alkyl chain incorporation, cyclization and peptide bond substitution, and where those strategies have been, or could be, applied to PET peptide-based radiopharmaceuticals.
Topics: Acylation; Animals; Carbon Radioisotopes; Cyclization; Fluorine Radioisotopes; Gallium Radioisotopes; Half-Life; Humans; Methylation; Peptides; Peptidomimetics; Positron-Emission Tomography; Protein Processing, Post-Translational; Protein Stability; Radiopharmaceuticals; Rodentia
PubMed: 32423178
DOI: 10.3390/molecules25102314 -
The Journal of Biological Chemistry Dec 2019Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes... (Review)
Review
Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive species, either purposely ( during pathogen killing or enzymatic reactions) or accidentally ( exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively-modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications-at the amino acid, peptide, or protein level-and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes.
Topics: Amino Acids; Animals; Anions; Antioxidants; Free Radicals; Humans; Kinetics; Nitric Oxide; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxygen; Peptides; Protein Processing, Post-Translational; Proteins; Reactive Oxygen Species; Ultraviolet Rays
PubMed: 31672919
DOI: 10.1074/jbc.REV119.006217