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Yakugaku Zasshi : Journal of the... Nov 2010L-Amino acid ligase (EC 6.3.2.28) is a microbial enzyme catalyzing formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner. The YwfE... (Review)
Review
L-Amino acid ligase (EC 6.3.2.28) is a microbial enzyme catalyzing formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner. The YwfE protein from Bacillus subtilis 168 was the first reported L-amino acid ligase, and it synthesizes various dipeptides. Thereafter, several L-amino acid ligases were newly obtained by in silico analysis using the ATP-grasp motif. But these L-amino acid ligases synthesize only dipeptide and no longer peptide. A novel L-amino acid ligase capable of catalyzing oligopeptide synthesis is required to increase the variety of peptides. We have previously found a new member of L-amino acid ligase, RizA, from B. subtilis NBRC3134, a microorganism that produces the peptide-antibiotic rhizocticin. We newly found that a gene at approximately 9 kbp upstream of rizA encoded a novel L-amino acid ligase RizB. Recombinant RizB synthesized homo-oligomers of branched-chain amino acids consisting of 2 to 5 amino acids, and also synthesized various heteropeptides. RizB is the first reported L-amino acid ligase that catalyzes oligopeptide synthesis. In addition, we obtained L-amino acid ligases showing oligopeptide synthesis activities by in silico analysis using BLAST, which is a set of similarity search programs. These L-amino acid ligases showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacteria adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones.
Topics: Amino Acids; Amino Acids, Branched-Chain; Bacillus subtilis; Biocatalysis; High-Throughput Screening Assays; Ligases; Oligopeptides; Organophosphorus Compounds; Recombinant Proteins
PubMed: 21048404
DOI: 10.1248/yakushi.130.1463 -
Amino Acids Sep 2016Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging...
Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging process. Amino acid racemization involves deprotonation of the alpha carbon and replacement of the proton in the opposite stereoconfiguration; this reaction is much faster for aspartate/asparagine than for other amino acids because these residues form a succinimide ring in which resonance stabilizes the carbanion resulting from proton loss. To determine if the replacement of the hydrogen atom on the alpha carbon with a deuterium atom might decrease the rate of racemization and thus stabilize polypeptides, we synthesized a hexapeptide, VYPNGA, in which the three carbon-bound protons in the asparaginyl residue were replaced with deuterium atoms. Upon incubation of this peptide in pH 7.4 buffer at 37 °C, we found that the rate of deamidation via the succinimide intermediate was unchanged by the presence of the deuterium atoms. However, the accumulation of the D-aspartyl and D-isoaspartyl-forms resulting from racemization and hydrolysis of the succinimide was decreased more than five-fold in the deuterated peptide over a 20 day incubation at physiological temperature and pH. Additionally, we found that the succinimide intermediate arising from the degradation of the deuterated asparaginyl peptide was slightly less likely to open to the isoaspartyl configuration than was the protonated succinimide. These findings suggest that the kinetic isotope effect resulting from the presence of deuteriums in asparagine residues can limit the accumulation of at least some of the degradation products that arise as peptides and proteins age.
Topics: Asparagine; Deuterium; Oligopeptides
PubMed: 27169868
DOI: 10.1007/s00726-016-2250-z -
Cancer Medicine Jan 2024RGD peptide can be found in cell adhesion and signaling proteins, such as fibronectin, vitronectin, and fibrinogen. RGD peptides' principal function is to facilitate... (Review)
Review
RGD peptide can be found in cell adhesion and signaling proteins, such as fibronectin, vitronectin, and fibrinogen. RGD peptides' principal function is to facilitate cell adhesion by interacting with integrin receptors on the cell surface. They have been intensively researched for use in biotechnology and medicine, including incorporation into biomaterials, conjugation to medicinal molecules or nanoparticles, and labeling with imaging agents. RGD peptides can be utilized to specifically target cancer cells and the tumor vasculature by engaging with these integrins, improving drug delivery efficiency and minimizing adverse effects on healthy tissues. RGD-functionalized drug carriers are a viable option for cancer therapy as this focused approach has demonstrated promise in the future. Writing a review on the RGD peptide can significantly influence how drugs are developed in the future by improving our understanding of the peptide, finding knowledge gaps, fostering innovation, and making drug design easier.
Topics: Humans; Oligopeptides; Peptides; Integrins; Neoplasms
PubMed: 38349028
DOI: 10.1002/cam4.6800 -
STAR Protocols Sep 2020This protocol describes immunoprecipitation of proteins associated with FLAG-tagged recombinant proteins followed by mass spectrometry-based proteomics to identify the...
This protocol describes immunoprecipitation of proteins associated with FLAG-tagged recombinant proteins followed by mass spectrometry-based proteomics to identify the associated interactome components. FLAG epitope was chosen, because existing high-affinity monoclonal antibodies allow for sensitive immunoprecipitation and FLAG peptides permit efficient elution of protein complexes. With many commercially available FLAG tools, this protocol is highly versatile. This procedure reduces immunoprecipitation of nonspecific binding proteins. Gene ontology analyses performed following mass spectrometry-based proteomics may elucidate novel functions of proteins of interest. For complete details on the use and application of this protocol, please refer to Valdez-Sinon et al. (2020).
Topics: Antibodies, Monoclonal; Epitopes; Immunoprecipitation; Oligopeptides; Recombinant Fusion Proteins; Recombinant Proteins
PubMed: 33111116
DOI: 10.1016/j.xpro.2020.100083 -
Journal of Visualized Experiments : JoVE May 2017Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions...
Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5' adenosine monophosphate-activated protein kinase (AMPK) has been established and was validated using a positional scanning peptide library assay. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.
Topics: AMP-Activated Protein Kinases; Binding Sites; Chromatography, Liquid; Mass Spectrometry; Oligopeptides; Peptide Library; Phosphorylation
PubMed: 28570513
DOI: 10.3791/55708 -
Nutrients Aug 2023, which encodes the monocarboxylate transporter 13 (MCT13), is a susceptibility gene for type 2 diabetes and is expressed in the liver and duodenum. Some...
, which encodes the monocarboxylate transporter 13 (MCT13), is a susceptibility gene for type 2 diabetes and is expressed in the liver and duodenum. Some peptidase-resistant oligopeptides are absorbed in the gastrointestinal tract and affect glycemic control in the body. Their efficient absorption is mediated by oligopeptide transporter(s) at the apical and basolateral membranes of the intestinal epithelia; however, the molecules responsible for basolateral oligopeptide transport have not been identified. In this study, we examined whether MCT13 functions as a novel basolateral oligopeptide transporter. We evaluated the uptake of oligopeptides and peptidomimetics in MCT13-transfected cells. The uptake of cephradine, a probe for peptide transport system(s), significantly increased in MCT13-transfected cells, and this increase was sensitive to membrane potential. The cellular accumulation of bioactive peptides, such as anserine and carnosine, was decreased by MCT13, indicating MCT13-mediated efflux transport activity. In polarized Caco-2 cells, MCT13 was localized at the basolateral membrane. MCT13 induction enhanced cephradine transport in an apical-to-basal direction across Caco-2 cells. These results indicate that MCT13 functions as a novel efflux transporter of oligopeptides and peptidomimetics, driven by electrochemical gradients across the plasma membrane, and it may be involved in the transport of these compounds across the intestinal epithelia.
Topics: Humans; Caco-2 Cells; Cephradine; Diabetes Mellitus, Type 2; Peptidomimetics; Cell Membrane; Oligopeptides
PubMed: 37630718
DOI: 10.3390/nu15163527 -
ChemMedChem Jul 2022Nanomedicine emerged some decades ago with the hope to be the solution for most unmet medical needs. However, tracking materials at nanoscale is challenging to their...
Nanomedicine emerged some decades ago with the hope to be the solution for most unmet medical needs. However, tracking materials at nanoscale is challenging to their reduced size, below the resolution limit of most conventional techniques. In this context, we propose the use of direct stochastic optical reconstruction microscopy (dSTORM) to study time stability and cell trafficking after transfection of oligopeptide end-modified poly(β-aminoester) (OM-pBAE) nanoparticles. We selected different combinations of cationic end oligopeptides (arginine - R; histidine - H; and lysine - K) among polymer libraries, since the oligopeptide combination demonstrated to be useful for different applications, such as vaccination and gene silencing. We demonstrate that their time evolution as well as their cell uptake and trafficking are dependent on the oligopeptide. This study opens the pave to broad mechanistic studies at nanoscale that could enable a rational selection of specific pBAE nanoparticles composition after determining their stability and cell trafficking.
Topics: Green Fluorescent Proteins; Microscopy; Nanoparticles; Oligopeptides; Transfection
PubMed: 35212466
DOI: 10.1002/cmdc.202100633 -
Biochimica Et Biophysica Acta Mar 2015Cellular uptake of small peptides is an important physiological process mediated by the PTR family of proton-coupled peptide transporters. In bacteria peptides can be... (Review)
Review
BACKGROUND
Cellular uptake of small peptides is an important physiological process mediated by the PTR family of proton-coupled peptide transporters. In bacteria peptides can be used as a source of amino acids and nitrogen. Similarly in humans peptide transport is the principle route for the uptake and retention of dietary protein in the form of short di- and tri-peptides for cellular metabolism.
SCOPE OF THE REVIEW
Recent crystal structures of bacterial PTR family transporters, combined with biochemical studies of transport have revealed key molecular details underpinning ligand promiscuity and the mechanism of proton-coupled transport within the family.
MAJOR CONCLUSIONS
Pairs of salt bridge interactions between transmembrane helices work in tandem to orchestrate alternating access transport within the PTR family. Key roles for residues conserved between bacterial and eukaryotic homologues suggest a conserved mechanism of peptide recognition and transport that in some cases has been subtly modified in individual species.
GENERAL SIGNIFICANCE
Physiological studies on PepT1 and PepT2, the mammalian members of this family, have identified these transporters as being responsible for the uptake of many pharmaceutically important drug molecules, including antibiotics and antiviral medications and demonstrated their promiscuity can be used for improving the oral bioavailability of poorly absorbed compounds. The insights gained from recent structural studies combined with previous physiological and biochemical analyses are rapidly advancing our understanding of this medically important transporter superfamily. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.
Topics: Amino Acid Sequence; Bacterial Proteins; Biological Transport; Membrane Transport Proteins; Models, Molecular; Molecular Sequence Data; Oligopeptides; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Homology, Amino Acid
PubMed: 24859687
DOI: 10.1016/j.bbagen.2014.05.011 -
Journal of Peptide Science : An... Apr 2017Several natural peptides have a biaryl or biaryl ether motif in their biologically active structures. A model bicyclic pentapeptide containing a biaryl bridge has been...
Several natural peptides have a biaryl or biaryl ether motif in their biologically active structures. A model bicyclic pentapeptide containing a biaryl bridge has been synthesized by solid-phase peptide synthesis combining on-resin Suzuki and Miyaura cross-coupling reactions. Its biological properties in terms of permeability, stability and cytotoxicity have been studied, demonstrating the positive contribution of the biaryl bridge, excellent membrane penetration and serum stability Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Topics: Biological Products; Cell Survival; Dose-Response Relationship, Drug; HeLa Cells; Humans; Molecular Conformation; Oligopeptides; Solid-Phase Synthesis Techniques; Structure-Activity Relationship
PubMed: 28303616
DOI: 10.1002/psc.2993 -
Molecular Pharmaceutics Dec 2017In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is...
In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is located across the apical membranes of the small intestine and kidney, where it serves as a high-capacity low-affinity transporter of a broad range of di- and tripeptides. hPepT1 is also overexpressed in the colon of inflammatory bowel disease (IBD) patients, where it mediates the transport of harmful peptides of bacterial origin. Therefore, hPepT1 is a drug target for prodrug substrates interacting with intracellular proteins or inhibitors blocking the transport of toxic bacterial products. In this study, we construct multiple structural models of hPepT1 representing different conformational states that occur during transport and inhibition. We then identify and characterize five ligands of hPepT1 using computational methods, such as virtual screening and QM-polarized ligand docking (QPLD), and experimental testing with uptake kinetic measurements and electrophysiological assays. Our results improve our understanding of the substrate and inhibitor specificity of hPepT1. Furthermore, the newly discovered ligands exhibit unique chemotypes, providing a framework for developing tool compounds with optimal intestinal absorption as well as future IBD therapeutics against this emerging drug target.
Topics: Biological Transport, Active; Drug Evaluation, Preclinical; Humans; Inhibitory Concentration 50; Intestinal Absorption; Kinetics; Ligands; Models, Chemical; Models, Molecular; Molecular Docking Simulation; Oligopeptides; Peptide Transporter 1; Prodrugs
PubMed: 29111754
DOI: 10.1021/acs.molpharmaceut.7b00775