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Methods in Enzymology 2021Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into...
Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into membrane proteins such as ion channels. Recently, this has increasingly been complemented by semi-synthetic strategies, in which part of the protein is generated synthetically. This means a vast range of chemical modifications, including non-canonical amino acids (ncAA), backbone modifications, chemical handles, fluorescent or spectroscopic labels and any combination of these can be incorporated. Among these approaches, protein trans-splicing (PTS) is particularly promising for protein reconstitution in live cells. It relies on one or more split inteins, which can spontaneously and covalently link flanking peptide or protein sequences. Here, we describe the use of PTS and its variant tandem PTS (tPTS) in semi-synthesis of ion channels in Xenopus laevis oocytes to incorporate ncAAs, post-translational modifications or metabolically stable mimics thereof. This strategy has the potential to expand the type and number of modifications in ion channel research.
Topics: Inteins; Ion Channels; Peptides; Protein Engineering; Protein Splicing; Trans-Splicing
PubMed: 34120713
DOI: 10.1016/bs.mie.2021.01.028 -
Journal of Structural Biology Dec 2023Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and...
Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in oxidative stress resistance, iron storage, and sulfur metabolism. Encapsulin shells exhibit icosahedral geometry and consist of 60, 180, or 240 identical protein subunits. Cargo encapsulation is mediated by the specific interaction of targeting peptides or domains, found in all cargo proteins, with the interior surface of the encapsulin shell during shell self-assembly. Here, we report the 2.53 Å cryo-EM structure of a heterologously produced and highly cargo-loaded T3 encapsulin shell from Myxococcus xanthus and explore the systems' structural heterogeneity. We find that exceedingly high cargo loading results in the formation of substantial amounts of distorted and aberrant shells, likely caused by a combination of unfavorable steric clashes of cargo proteins and shell conformational changes. Based on our cryo-EM structure, we determine and analyze the targeting peptide-shell binding mode. We find that both ionic and hydrophobic interactions mediate targeting peptide binding. Our results will guide future attempts at rationally engineering encapsulins for biomedical and biotechnological applications.
Topics: Bacterial Proteins; Bacteria; Oxidative Stress; Archaea; Peptides
PubMed: 37657675
DOI: 10.1016/j.jsb.2023.108022 -
Nature Jun 2020Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of... (Comparative Study)
Comparative Study
Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported, advances in mass-spectrometry-based proteomics have enabled increasingly comprehensive identification and quantification of the human proteome. However, there have been few comparisons across species, in stark contrast with genomics initiatives. Here we use an advanced proteomics workflow-in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system-for the in-depth study of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to the scientific community. The data also provide a large-scale case study for sequence-based machine learning, as we demonstrate by experimentally confirming the predicted properties of peptides from Bacteroides uniformis. Our results offer a comparative view of the functional organization of organisms across the entire evolutionary range. A remarkably high fraction of the total proteome mass in all kingdoms is dedicated to protein homeostasis and folding, highlighting the biological challenge of maintaining protein structure in all branches of life. Likewise, a universally high fraction is involved in supplying energy resources, although these pathways range from photosynthesis through iron sulfur metabolism to carbohydrate metabolism. Generally, however, proteins and proteomes are remarkably diverse between organisms, and they can readily be explored and functionally compared at www.proteomesoflife.org.
Topics: Animals; Bacteroides; Carbohydrate Metabolism; Chromatography; Classification; Deep Learning; Glycolysis; Homeostasis; Ion Transport; Iron-Sulfur Proteins; Oxidation-Reduction; Peptides; Photosynthesis; Protein Biosynthesis; Protein Folding; Proteolysis; Proteome; Proteomics; Species Specificity
PubMed: 32555458
DOI: 10.1038/s41586-020-2402-x -
Nature Chemical Biology May 2024Cyclic peptides can bind challenging disease targets with high affinity and specificity, offering enormous opportunities for addressing unmet medical needs. However, as...
Cyclic peptides can bind challenging disease targets with high affinity and specificity, offering enormous opportunities for addressing unmet medical needs. However, as with biological drugs, most cyclic peptides cannot be applied orally because they are rapidly digested and/or display low absorption in the gastrointestinal tract, hampering their development as therapeutics. In this study, we developed a combinatorial synthesis and screening approach based on sequential cyclization and one-pot peptide acylation and screening, with the possibility of simultaneously interrogating activity and permeability. In a proof of concept, we synthesized a library of 8,448 cyclic peptides and screened them against the disease target thrombin. Our workflow allowed multiple iterative cycles of library synthesis and yielded cyclic peptides with nanomolar affinities, high stabilities and an oral bioavailability (%F) as high as 18% in rats. This method for generating orally available peptides is general and provides a promising push toward unlocking the full potential of peptides as therapeutics.
Topics: Peptides, Cyclic; Administration, Oral; Biological Availability; Animals; Rats; Humans; Cyclization; Peptide Library; Thrombin; Male; Combinatorial Chemistry Techniques; Acylation
PubMed: 38155304
DOI: 10.1038/s41589-023-01496-y -
Natural Product Reports Mar 2022The first machineries for non-ribosomal peptide (NRP) biosynthesis were uncovered over 50 years ago, and the dissection of these megasynthetases set the stage for the... (Review)
Review
The first machineries for non-ribosomal peptide (NRP) biosynthesis were uncovered over 50 years ago, and the dissection of these megasynthetases set the stage for the nomenclature system that has been used ever since. Although the number of exceptions to the canonical biosynthetic pathways has surged in the intervening years, the NRP synthetase (NRPS) classification system has remained relatively unchanged. This has led to the exclusion of many biosynthetic pathways whose biosynthetic machineries violate the classical rules for NRP assembly, and ultimately to a rupture in the field of NRP biosynthesis. In an attempt to unify the classification of NRP pathways and to facilitate the communication within the research field, we propose a revised framework for grouping ribosome-independent peptide biosynthetic pathways based on recognizable commonalities in their biosynthetic logic. Importantly, the framework can be further refined as needed.
Topics: Biosynthetic Pathways; Peptide Biosynthesis, Nucleic Acid-Independent; Peptide Synthases; Peptides; Ribosomes
PubMed: 34586117
DOI: 10.1039/d1np00019e -
ChemMedChem Jun 2022Pin1 catalyzes the cis-trans isomerization of pThr-Pro or pSer-Pro amide bonds of various proteins involved in several physio/pathological processes. In this framework,...
Pin1 catalyzes the cis-trans isomerization of pThr-Pro or pSer-Pro amide bonds of various proteins involved in several physio/pathological processes. In this framework, recent research activity is directed toward the identification of new selective Pin1 inhibitors. Here, we developed a set of peptide-based Pin1 inhibitors. Direct-binding experiments allowed the identification of the peptide-based inhibitor 5 k (methylacetyl-l-alanyl-l-histidyl-l-prolyl-l-phenylalaninate) as a potent ligand of Pin1. Notably, 5 k binds Pin1 with higher affinity than Pin4. The comparative analysis of molecular models of Pin1 and Pin4 with the selected compound gave a rational explanation of the biochemical activity and pinpointed the chemical elements that, if opportunely modified, may further improve inhibitory potency, pharmacological properties, and selectivity of future peptide-based parvulin inhibitors. Since 5 k showed limited cell penetration and no antiproliferative activity, it was conjugated to a polyarginine stretch (R8), known to promote cell penetration of peptides, to obtain the R8-5 k derivative, which displayed antiproliferative effects on cancer cell lines over non-tumor cells. The effect of R8 on cell proliferation was also investigated. This work warrants caution about applying the R8 strategy in the development of cell-penetrating antiproliferative peptides, as it is not inert.
Topics: Models, Molecular; NIMA-Interacting Peptidylprolyl Isomerase; Peptides; Peptidylprolyl Isomerase; Phosphorylation
PubMed: 35357776
DOI: 10.1002/cmdc.202200050 -
Peptides Dec 2021The continued use of antibiotics has been accompanied by the rapid emergence and spread of antibiotic-resistant strains of bacteria. Antimicrobial peptides (AMPs), also... (Review)
Review
The continued use of antibiotics has been accompanied by the rapid emergence and spread of antibiotic-resistant strains of bacteria. Antimicrobial peptides (AMPs), also known as host defense peptides, show multiple features as an ideal antimicrobial agent, including potent, rapid, and broad-spectrum antimicrobial activity, low promotion of antimicrobial resistance, potent anti-biofilm activity, and lethality against metabolically inactive microorganisms. However, several crucial drawbacks constrain the use of AMPs as clinical drugs, e.g., liability in vivo, toxicity when used systemically, and high production costs. Based on recent findings and our own experiences, here we summarize some chemical modifications and key design strategies to increase the therapeutic potential of AMPs, including 1) enhancing antimicrobial activities, 2) improving in vivo effectiveness, and 3) reduction in toxicity, which may facilitate the design and optimization of AMPs for the development of drug candidates. We also discuss the present challenges in the optimization of AMPs and future concerns about the resistance and cross-resistance to AMPs in the development of AMPs as therapeutic drugs.
Topics: Antimicrobial Peptides; Cyclization; Humans; Microbial Sensitivity Tests; Nanoparticles; Protein Stability; Structure-Activity Relationship
PubMed: 34600037
DOI: 10.1016/j.peptides.2021.170666 -
Methods in Molecular Biology (Clifton,... 2020The application of designer peptides in medicinal chemistry, chemical biology, and materials science has generated new interest in synthetic methods for the structural...
The application of designer peptides in medicinal chemistry, chemical biology, and materials science has generated new interest in synthetic methods for the structural modification of amino acids. Strategies which facilitate the direct diversification of proteinogenic functional groups within unprotected peptide substrates are particularly attractive as they leverage modern solution- and solid-phase protocols-tools which are now both robust and routine-for the synthesis of native peptides. Accordingly, a recent approach to the decarboxylative functionalization of peptidic carboxylic acids, including aspartic/glutamic acid residues and α-carboxylic acids, has proven to be a promising new strategy for peptide modification. This synthetic method merges conventional strategies for the activation of carboxylic acids with transition metal-catalyzed cross-coupling chemistry to forge new C-C bonds for the late-stage introduction of valuable synthetic handles. This chapter details a step-by-step protocol for the activation and nickel-catalyzed decarboxylative alkylation of a simple peptide substrate to highlight the broad utility of this strategy for the synthesis of designer peptides.
Topics: Amino Acids; Carboxylic Acids; Decarboxylation; Ligands; Metals; Nickel; Oxidative Coupling; Peptides; Solid-Phase Synthesis Techniques
PubMed: 31879933
DOI: 10.1007/978-1-0716-0227-0_19 -
Nature Communications Sep 2021Peptide-protein interactions are involved in various fundamental cellular functions and their identification is crucial for designing efficacious peptide therapeutics....
Peptide-protein interactions are involved in various fundamental cellular functions and their identification is crucial for designing efficacious peptide therapeutics. Recently, a number of computational methods have been developed to predict peptide-protein interactions. However, most of the existing prediction approaches heavily depend on high-resolution structure data. Here, we present a deep learning framework for multi-level peptide-protein interaction prediction, called CAMP, including binary peptide-protein interaction prediction and corresponding peptide binding residue identification. Comprehensive evaluation demonstrated that CAMP can successfully capture the binary interactions between peptides and proteins and identify the binding residues along the peptides involved in the interactions. In addition, CAMP outperformed other state-of-the-art methods on binary peptide-protein interaction prediction. CAMP can serve as a useful tool in peptide-protein interaction prediction and identification of important binding residues in the peptides, which can thus facilitate the peptide drug discovery process.
Topics: Algorithms; Binding Sites; Computational Biology; Deep Learning; Models, Molecular; Peptides; Protein Binding; Protein Domains; Proteins; Reproducibility of Results
PubMed: 34526500
DOI: 10.1038/s41467-021-25772-4 -
ChemMedChem Jul 2020Among the many methods available for accessing conformationally diverse cyclic peptides, the derivatization of macrocyclic iminopeptides has remained notably... (Review)
Review
Among the many methods available for accessing conformationally diverse cyclic peptides, the derivatization of macrocyclic iminopeptides has remained notably underexplored. Now, a relevant complexity-generating method expands the repertoire of synthetic strategies exploiting the reactivity of an imino bond embedded in the cyclic peptide skeleton. Here we highlight a recent report describing the on-resin construction of a new family of macrocyclic peptide/natural product-inspired hybrids, namely "PepNats", by derivatization of cyclic iminopeptides through 1,3-cycloaddition reactions. A proof-of-concept with PepNats bearing peptide sequences that mimic protein hot loops demonstrated the potential of this strategy to create novel macrocyclic peptide ligands capable of modulating protein-protein interactions.
Topics: Biological Products; Imines; Ligands; Macrocyclic Compounds; Molecular Conformation; Peptides; Protein Binding; Proteins
PubMed: 32516501
DOI: 10.1002/cmdc.202000261