Discovery of membrane-permeating cyclic peptides via mRNA display

AbstractSmall synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and non-disruptively controlling intracellular or intraembryonic processes.

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Versatile proteome labelling in fruit flies with SILAF

10.1101/590315 ◽

2019 ◽

Author(s):

Florian A. Schober ◽

Ilian Atanassov ◽

David Moore ◽

Anna Wedell ◽

Christoph Freyer ◽

...

Keyword(s):

Protein Turnover ◽

Cell Biology ◽

Metabolic Regulation ◽

Developmental Stages ◽

Fruit Fly ◽

Phosphorylation Sites ◽

Turnover Rates ◽

Post Translational Modification ◽

Stable Isotope Labelling ◽

Rapid Generation

ABSTRACTDrosophila melanogaster has been a working horse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to technical obstacles, especially the lack of reliable labelling methods. Here, we advanced a chemically defined food source into stable-isotope labelling of amino acids in flies (SILAF). It allows for the rapid generation of a large number of flies with full incorporation of lysine-6. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 5,966 proteins and 7,496 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. Furthermore, the label can be traced and predicts protein turnover rates during different developmental stages. The ease and versatility of the method actuates the fruit fly as an appealing model in proteomic and post-translational modification studies and it enlarges potential metabolic applications based on heavy amino acid diets.

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Gene targeting, principles, and practice in mammalian cells

Gene Targeting ◽

10.1093/oso/9780199637928.003.0005 ◽

1999 ◽

Author(s):

Paul Hasty ◽

Alejandro Abuin

Keyword(s):

Gene Targeting ◽

Mammalian Cells ◽

Es Cells ◽

Globin Gene ◽

Marker Gene ◽

Embryonic Stem ◽

Fibroblast Cell ◽

Chromosomal Locus ◽

Target Locus

When a fragment of genomic DNA is introduced into a mammalian cell it can locate and recombine with the endogenous homologous sequences. This type of homologous recombination, known as gene targeting, is the subject of this chapter. Gene targeting has been widely used, particularly in mouse embryonic stem (ES) cells, to make a variety of mutations in many different loci so that the phenotypic consequences of specific genetic modifications can be assessed in the organism. The first experimental evidence for the occurrence of gene targeting in mammalian cells was made using a fibroblast cell line with a selectable artificial locus by Lin et al. (1), and was subsequently demonstrated to occur at the endogenous β-globin gene by Smithies et al. in erythroleukaemia cells (2). In general, the frequencies of gene targeting in mammalian cells are relatively low compared to yeast cells and this is probably related to, at least in part, a competing pathway: efficient integration of the transfected DNA into a random chromosomal site. The relative ratio of targeted to random integration events will determine the ease with which targeted clones are identified in a gene targeting experiment. This chapter details aspects of vector design which can determine the efficiency of recombination, the type of mutation that may be generated in the target locus, as well as the selection and screening strategies which can be used to identify clones of ES cells with the desired targeted modification. Since the most common experimental strategy is to ablate the function of a target gene (null allele) by introducing a selectable marker gene, we initially describe the vectors and the selection schemes which are helpful in the identification of recombinant clones (Sections 2-5). In Section 6, we describe the vectors and additional considerations for generating subtle mutations in a target locus devoid of any exogenous sequences. Finally, Section 7 is dedicated to the use of gene targeting as a method to express exogenous genes from specific endogenous regulatory elements in vivo, also known as ‘knock-in’ strategies. A targeting vector is designed to recombine with and mutate a specific chromosomal locus.

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Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display

10.1101/2021.08.23.457419 ◽

2021 ◽

Author(s):

Jason Johansen-Leete ◽

Sven Ullrich ◽

Sarah Fry ◽

Rebecca Frkic ◽

Max Bedding ◽

...

Keyword(s):

Cyclic Peptides ◽

Cyclic Peptide ◽

Peptide Inhibitors ◽

Mrna Display ◽

High Affinity ◽

Main Protease ◽

Binding Epitope ◽

Affinity Peptide ◽

Micromolar Range

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

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A Target-Based Method for Designing Heterochiral Cyclic Peptide Binders: De Novo Inhibitors of the PD-1/PD-L1 Interaction

10.26434/chemrxiv.11663337.v2 ◽

2020 ◽

Author(s):

Salvador Guardiola ◽

Monica Varese ◽

Xavier Roig ◽

Jesús Garcia ◽

Ernest Giralt

Keyword(s):

Protein Interactions ◽

Cyclic Peptides ◽

General Framework ◽

Large Scale ◽

Cyclic Peptide ◽

De Novo ◽

Inhibitory Effect ◽

Original Text ◽

Retraction Notice ◽

Pharmaceutical Properties

NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above. ------------------------------------------------------------------------ Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-i3 and PD-i6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our de novo design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.

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Basic Biology of Trypanosoma Cruzi

Current Pharmaceutical Design ◽

10.2174/1381612826999201203213527 ◽

2020 ◽

Vol 26 ◽

Author(s):

Aline Araujo Zuma ◽

Emile dos Santos Barrias ◽

Wanderley de Souza

Keyword(s):

Trypanosoma Cruzi ◽

Trypanosoma Brucei ◽

Cell Biology ◽

Structural Organization ◽

Developmental Stages ◽

Endocytic Pathway ◽

Host Cells ◽

Cellular Organization ◽

Basic Biology ◽

Comparative Information

Abstract:: The present review addresses basic aspects of the biology of the pathogenic protozoa Trypanosoma cruzi and some comparative information with Trypanosoma brucei. Like eukaryotic cells, their cellular organization is similar to that of mammalian hosts. However, these parasites present structural particularities. That is why the following topics are emphasized in this paper: developmental stages of the life cycle in the vertebrate and invertebrate hosts; the cytoskeleton of the protozoa, especially the sub-pellicular microtubules; the flagellum and its attachment to the protozoan body through specialized junctions; the kinetoplast-mitochondrion complex, including its structural organization and DNA replication; the glycosome and its role in the metabolism of the cell; the acidocalcisome, describing its morphology, biochemistry, and functional role; the cytostome and the endocytic pathway; the organization of the endoplasmic reticulum and Golgi complex; the nucleus, describing its structural organization during interphase and division; and the process of interaction of the parasite with host cells. The unique characteristics of these structures also make them interesting chemotherapeutic targets. Therefore, further understanding of cell biology aspects contributes to the development of drugs for chemotherapy.

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Potent Cyclic Peptide Inhibitors of FXIIa Discovered by mRNA Display with Genetic Code Reprogramming

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.1c00651 ◽

2021 ◽

Author(s):

Daniel J. Ford ◽

Nisharnthi M. Duggan ◽

Sarah E. Fry ◽

Jorge Ripoll-Rozada ◽

Stijn M. Agten ◽

...

Keyword(s):

Genetic Code ◽

Cyclic Peptide ◽

Peptide Inhibitors ◽

Mrna Display

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Wnt/β-catenin Pathway-Mediated PPARδ Expression during Embryonic Development Differentiation and Disease

International Journal of Molecular Sciences ◽

10.3390/ijms22041854 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1854

Author(s):

Tabinda Sidrat ◽

Zia-Ur Rehman ◽

Myeong-Don Joo ◽

Kyeong-Lim Lee ◽

Il-Keun Kong

Keyword(s):

Embryonic Development ◽

Transcriptional Activation ◽

Target Gene ◽

Developmental Stages ◽

Embryonic Stem ◽

Hereditary Diseases ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Stem Cell Regulation ◽

Early Developmental

The Wnt/β-catenin signaling pathway plays a crucial role in early embryonic development. Wnt/β-catenin signaling is a major regulator of cell proliferation and keeps embryonic stem cells (ESCs) in the pluripotent state. Dysregulation of Wnt signaling in the early developmental stages causes several hereditary diseases that lead to embryonic abnormalities. Several other signaling molecules are directly or indirectly activated in response to Wnt/β-catenin stimulation. The crosstalk of these signaling factors either synergizes or opposes the transcriptional activation of β-catenin/Tcf4-mediated target gene expression. Recently, the crosstalk between the peroxisome proliferator-activated receptor delta (PPARδ), which belongs to the steroid superfamily, and Wnt/β-catenin signaling has been reported to take place during several aspects of embryonic development. However, numerous questions need to be answered regarding the function and regulation of PPARδ in coordination with the Wnt/β-catenin pathway. Here, we have summarized the functional activation of the PPARδ in co-ordination with the Wnt/β-catenin pathway during the regulation of several aspects of embryonic development, stem cell regulation and maintenance, as well as during the progression of several metabolic disorders.

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Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients

AJP Cell Physiology ◽

10.1152/ajpcell.00188.2019 ◽

2019 ◽

Vol 317 (4) ◽

pp. C725-C736

Author(s):

Gurbind Singh ◽

Divya Sridharan ◽

Mahmood Khan ◽

Polani B. Seshagiri

Keyword(s):

Cell Biology ◽

Fluorescent Protein ◽

Es Cells ◽

Monochromatic Light ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Medical Diagnostics ◽

Calcium Transients ◽

Egfp Expression ◽

Mitochondrial Activity

We earlier established the mouse embryonic stem (ES) cell “GS-2” line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420–495), green (~510–575), and red (~600–700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

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CALINCA—A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease

Cells ◽

10.3390/cells10030692 ◽

2021 ◽

Vol 10 (3) ◽

pp. 692

Author(s):

Sweta Talyan ◽

Samantha Filipów ◽

Michael Ignarski ◽

Magdalena Smieszek ◽

He Chen ◽

...

Keyword(s):

Cell Biology ◽

Mammalian Cells ◽

De Novo ◽

Depth Information ◽

Gene Products ◽

Classical Analysis ◽

Protein Coding ◽

Bioinformatic Pipeline ◽

Non Coding Rnas ◽

Filtration Unit

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

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Improvement on Permeability of Cyclic Peptide/Peptidomimetic: Backbone N-Methylation as A Useful Tool

Marine Drugs ◽

10.3390/md19060311 ◽

2021 ◽

Vol 19 (6) ◽

pp. 311

Author(s):

Yang Li ◽

Wang Li ◽

Zhengshuang Xu

Keyword(s):

Cyclic Peptides ◽

Cyclic Peptide ◽

Three Dimensional ◽

Conformational Space ◽

Cell Permeability ◽

Relative Importance ◽

Intrinsic Properties ◽

Surface Areas ◽

Synthetic Methodologies ◽

Three Dimensional Configuration

Peptides have a three-dimensional configuration that can adopt particular conformations for binding to proteins, which are well suited to interact with larger contact surface areas on target proteins. However, low cell permeability is a major challenge in the development of peptide-related drugs. In recent years, backbone N-methylation has been a useful tool for manipulating the permeability of cyclic peptides/peptidomimetics. Backbone N-methylation permits the adjustment of molecule’s conformational space. Several pathways are involved in the drug absorption pathway; the relative importance of each N-methylation to total permeation is likely to differ with intrinsic properties of cyclic peptide/peptidomimetic. Recent studies on the permeability of cyclic peptides/peptidomimetics using the backbone N-methylation strategy and synthetic methodologies will be presented in this review.

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