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Hormone and Metabolic Research =... 2004A role for glucagon-like peptide 1 (GLP-1) has been suggested in stimulating beta-cell lipolysis via elevation of cAMP and activation of protein kinase A, which in turn...
A role for glucagon-like peptide 1 (GLP-1) has been suggested in stimulating beta-cell lipolysis via elevation of cAMP and activation of protein kinase A, which in turn may activate hormone-sensitive lipase (HSL), thereby contributing to fatty acid generation (FFA) from intracellular triglyceride stores. FFAs may then be metabolized to a lipid signal, which is required for optimal glucose-stimulated insulin secretion. Since HSL is expressed in islet beta-cells, this effect could contribute to the stimulation of insulin secretion by GLP-1, provided that a lipid signal of importance for insulin secretion is generated. To examine this hypothesis, we have studied the acute effect of GLP-1 on isolated mouse islets from normal mice and from mice with high-fat diet induced insulin resistance. We found, however, that although GLP-1 (100 nM) markedly potentiated glucose-stimulated insulin secretion from islets of both feeding groups, the peptide was not able to stimulate islet palmitate oxidation or increase lipolysis measured as glycerol release. This indicates that a lipid signal does not contribute to the acute stimulation of insulin secretion by GLP-1. To test whether lipolysis might be involved in the islet effects of long-term GLP-1 action, mice from the two feeding groups were chronically treated with exendin-4, a peptide that lowers blood glucose by interacting with GLP-1 receptors, in order to stimulate insulin secretion, for 16 days before isolation of the islets. The insulinotropic effects of GLP-1 and forskolin were exaggerated in isolated islets from exendin-4 treated mice given a high-fat diet, with a augmented palmitate oxidation as well as islet lipolysis at high glucose levels in these islets. Exendin-4 treatment had less impact on mice fed a normal diet. From these results we conclude that while GLP-1 does not seem to induce beta-cell lipolysis acutely in mouse islets, the peptide affects beta-cell fat metabolism after long-term adaptation to GLP-1 receptor stimulation.
Topics: Animals; Diabetes Mellitus, Type 2; Disease Models, Animal; Exenatide; Fatty Acids, Nonesterified; Female; Glucagon; Glucagon-Like Peptide 1; Glucose; Glucose Tolerance Test; Hypoglycemic Agents; Insulin; Insulin Secretion; Islets of Langerhans; Lipid Metabolism; Lipolysis; Mice; Mice, Inbred C57BL; Peptide Fragments; Peptides; Protein Precursors; Venoms
PubMed: 15655711
DOI: 10.1055/s-2004-826166 -
Drug Discovery Today. Technologies Dec 2017Constrained peptides pose tremendous value in drug discovery. For example, owing to their large surface areas, they offer novel ways at inhibiting protein-protein... (Review)
Review
Constrained peptides pose tremendous value in drug discovery. For example, owing to their large surface areas, they offer novel ways at inhibiting protein-protein interactions. As this field has grown, it has become desirable to introduce non-peptidic functionality into these rings to enable differentiated structure activity relationships and improved pharmacokinetic properties. Recent advances in the synthesis of cyclic pseudopeptides include macrocyclization through cysteine alkylation, multicomponent reactions, decarboxylative couplings, and novel stapling chemistry.
Topics: Alkylation; Carbon; Cyclization; Cysteine; Peptides, Cyclic
PubMed: 29249240
DOI: 10.1016/j.ddtec.2017.11.004 -
Molecular Metabolism Oct 2023Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic...
OBJECTIVE
Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed.
METHODS
We developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R→A).
RESULTS
We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by an environmental temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue.
CONCLUSIONS
The ΔTLQP-21 mouse line can be a valuable resource to conduct mechanistic studies on the necessary role of TLQP-21 in physiology and disease, while also serving as a platform to test the specificity of novel antibodies or immunoassays directed at TLQP-21. Our approach also has far-reaching implications by informing the development of knowledge-based genetic engineering approaches to generate selective loss of function of other peptides encoded by pro-hormones genes, leaving all other peptides within the pro-protein precursor intact and unmodified.
Topics: Animals; Mice; Diet; Homeostasis; Neuropeptides; Peptide Fragments; Peptide Hormones; Energy Metabolism
PubMed: 37482186
DOI: 10.1016/j.molmet.2023.101781 -
Journal of Pharmaceutical Sciences Sep 2012Previously, our laboratory reported that cyclic peptide prodrugs of the opioid peptide H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) are metabolized by cytochrome P450 (CYP450)...
Factors that restrict the cell permeation of cyclic prodrugs of an opioid peptide, part 3: Synthesis of analogs designed to have improved stability to oxidative metabolism.
Previously, our laboratory reported that cyclic peptide prodrugs of the opioid peptide H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) are metabolized by cytochrome P450 (CYP450) enzymes, which limits their systemic exposure after oral dosing to animals. In an attempt to design more metabolically stable cyclic prodrugs of DADLE, we synthesized analogs of DADLE cyclized with a coumarinic acid linker (CA; CA-DADLE), which contained modifications in the amino acid residues known to be susceptible to CYP450 oxidation. Metabolic stability and metabolite identification studies of CA-DADLE and its analogs were then compared using rat liver microsomes (RLM), guinea pig liver microsomes (GPLM), and human liver microsomes (HLM), as well as recombinant human recombinant cytochrome P450 3A4 (hCYP3A4). Similar to the results observed for CA-DADLE, incubation of its analogs with RLM, GPLM, and HLM resulted in monohydroxylation of an amino acid side chain on these cyclic prodrugs. When CA-DADLE was incubated with hCYP3A4, similar oxidative metabolism of the peptide was observed. In contrast, incubation of the CA-DADLE analogs with hCYP3A4 showed that these amino-acid-modified analogs are not substrates for this CYP450 isozyme. These results suggest that the amino-acid-modified analogs of CA-DADLE prepared in this study could be stable to metabolic oxidation by CYP3A4 expressed in human intestinal mucosal cells.
Topics: Analgesics, Opioid; Animals; Biotransformation; Cell Membrane Permeability; Chemistry, Pharmaceutical; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Drug Design; Drug Stability; Enkephalin, Leucine-2-Alanine; Female; Guinea Pigs; Humans; Hydroxylation; Male; Microsomes, Liver; Oxidation-Reduction; Prodrugs; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Substrate Specificity; Technology, Pharmaceutical
PubMed: 22411763
DOI: 10.1002/jps.23109 -
Methods in Molecular Biology (Clifton,... 2016The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide...
The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide libraries on cellulose membrane supports. The synthesis method is based on conventional Fmoc chemistry building up peptides with free N-terminal amino acids starting at their cellulose-coupled C-termini. Several hundreds of peptide sequences can be assembled with this technique on one membrane comprising a strong binding potential due to high local peptide concentrations. Peptide orientation on SPOT membranes qualifies this array type for assaying substrate specificities of N-recognins, the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Pioneer studies described binding capability of mammalian and yeast enzymes depending on a peptide's N-terminus. SPOT arrays have been successfully used to describe substrate specificity of N-recognins which are the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Here, we describe the implementation of SPOT binding assays with focus on the identification of N-recognin substrates, applicable also for plant NERD enzymes.
Topics: Amino Acid Sequence; Cellulose; Molecular Biology; Neoplasm Proteins; Peptide Library; Peptides; Protein Array Analysis; Protein Binding; Substrate Specificity; Ubiquitin-Protein Ligases
PubMed: 27424747
DOI: 10.1007/978-1-4939-3759-2_7 -
Methods in Molecular Biology (Clifton,... 2019Peptides provide an attractive modality for targeting challenging drug targets such as intracellular protein-protein interactions. Unfortunately, peptides are generally...
Peptides provide an attractive modality for targeting challenging drug targets such as intracellular protein-protein interactions. Unfortunately, peptides are generally impermeable to the cell membrane and inherently susceptible to proteolytic degradation in vivo. Macrocyclization of peptides greatly increases their proteolytic stability and in some cases the cell-penetrating activity. Conjugation of peptidyl cargoes to cyclic cell-penetrating peptides has resulted in potent, cell-permeable, and metabolically stable macrocyclic peptides against intracellular protein targets. Proper conjugation/integration of a peptidyl cargo with a cyclic cell-penetrating peptide is critical to retain the activity of each component and generate a biologically active macrocyclic peptide. This chapter describes the different conjugation strategies that have been developed (including endocyclic, bicyclic, and reversible cyclization methods) and the detailed protocols for their preparation.
Topics: Amino Acid Sequence; Cell Membrane Permeability; Cell-Penetrating Peptides; Cyclization; Drug Delivery Systems; HeLa Cells; Humans; Peptides, Cyclic; Proteolysis
PubMed: 31134566
DOI: 10.1007/978-1-4939-9504-2_3 -
Current Pharmaceutical Design 2013Peptides are important signaling modules, acting both as individual hormones and as parts of larger molecules, mediating their protein-protein interactions. Many... (Review)
Review
Peptides are important signaling modules, acting both as individual hormones and as parts of larger molecules, mediating their protein-protein interactions. Many peptidic and peptidomimetic drugs have reached the marketplace and opportunities for peptide-based drug discovery are on the rise. pH-dependent behavior of peptides is well documented in the context of misfolding diseases and peptide translocation. Changes in the protonation states of peptide residues often have a crucial effect on a peptide's structure, dynamics and function, which may be exploited for biotechnological applications. The current review surveys the increasing levels of sophistication in the treatment of protonation states in computational studies involving peptides. Specifically we describe I) the common practice of assigning a single protonation state and using it throughout the dynamic simulation, II) approaches that consider multiple protonation states and compare computed observables to experimental ones, III) constant pH molecular dynamics methods that couple changes in protonation states with conformational dynamics "on the fly". Applications of conformational dynamics treatment of peptides in the context of binding, folding and interactions with the membrane are presented, illustrating the growing body of work in this field and highlighting the importance of careful handling of protonation states of peptidic residues.
Topics: Molecular Dynamics Simulation; Peptides; Peptidomimetics; Protein Binding; Protein Folding; Proteins; Protons
PubMed: 23170889
DOI: 10.2174/1381612811319230003 -
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 -
Pharmaceutical Research Oct 1995The study of peptide transport across the blood-brain barrier (BBB) is a field fraught with conflicting interpretations. This review presents a fairly strong case that... (Review)
Review
The study of peptide transport across the blood-brain barrier (BBB) is a field fraught with conflicting interpretations. This review presents a fairly strong case that peptides can be differentially transported at the BBB. However, minimal transport of peptides could have important impact on central nervous system (CNS) functions since only small amounts are needed for physiologic pharmacologic and/or pathologic effects. Several BBB peptide transport mechanisms (i.e., receptor-mediated, absorptive-mediated, carrier-mediated and non-specific passive diffusion), as well as non-transport processes (i.e., endocytosis without transcytosis, absorption and metabolism) are discussed. It is emphasized that peptide transport systems at the BBB could be important targets for both therapeutic delivery of peptides and the development of certain brain pathologies. Strategies to manipulate peptide BBB transport processes have been discussed including lipidization, chemical modifications of the N-terminal end, coupling of transport with post-BBB metabolism and formation of potent neuroactive peptides, up-regulation of putative peptide transporters, use of chimeric peptides in which non-transportable peptide is chemically linked to a transportable peptide, use of monoclonal antibodies against peptide receptors, and binding of circulating peptides to apolipoproteins. It is suggested that future directions should be directed towards development of molecular strategies to up-regulate specific BBB peptide transporters to enhance brain delivery of peptide neuropharmaceuticals, or to down-regulate transport of peptides with potential role in cerebral pathogenesis.
Topics: Animals; Biological Transport; Blood-Brain Barrier; Capillary Permeability; Cerebrovascular Circulation; Glutathione; Peptides
PubMed: 8584471
DOI: 10.1023/a:1016254514167 -
Journal of the American Chemical Society Feb 2023Macrocyclization improves the pharmaceutical properties of peptides; however, regio- and chemoselective intramolecular cyclizations remain challenging. Here we developed...
Macrocyclization improves the pharmaceutical properties of peptides; however, regio- and chemoselective intramolecular cyclizations remain challenging. Here we developed a streamlined chemoenzymatic approach to synthesize cyclic peptides by exploiting non-ribosomal peptide (NRP) cyclases. Linear peptides linked to the resin through a -terminal diol ester functionality are synthesized on a solid support, to circumvent the installation of leaving groups to the peptidic substrates in the liquid phase which often triggers undesirable epimerization. Cleavage of the resin-bound peptides yielded the diol esters with sufficient purity to be readily cyclized in a head-to-tail manner by SurE, a representative penicillin-binding protein-type thioesterase (PBP-type TE). Explorations of homologous wild-type enzymes as well as rational protein engineering have broadened the scope of the enzymatic macrolactamization. This method will potentially accelerate the exploitation of NRP cyclases as biocatalysts.
Topics: Peptides, Cyclic; Peptides; Cyclization
PubMed: 36638272
DOI: 10.1021/jacs.2c11082