Backbone-cyclized Polypeptides Research Articles

A cysteine selenosulfide redox switch for protein chemical synthesis

The control of cysteine reactivity is of paramount importance for the synthesis of proteins using the native chemical ligation (NCL) reaction. We report that this goal can be achieved in a traceless manner during ligation by appending a simple N-selenoethyl group to cysteine. While in synthetic organic chemistry the cleavage of carbon-nitrogen bonds is notoriously difficult, we describe that N-selenoethyl cysteine (SetCys) loses its selenoethyl arm in water under mild conditions upon reduction of its selenosulfide bond. Detailed mechanistic investigations show that the cleavage of the selenoethyl arm proceeds through an anionic mechanism with assistance of the cysteine thiol group. The implementation of the SetCys unit in a process enabling the modular and straightforward assembly of linear or backbone cyclized polypeptides is illustrated by the synthesis of biologically active cyclic hepatocyte growth factor variants.

Biotechnological Applications of Protein Splicing.

Protein splicing domains, also called inteins, have become a powerful biotechnological tool for applications involving molecular biology and protein engineering. Early applications of inteins focused on self-cleaving affinity tags, generation of recombinant polypeptide α-thioesters for the production of semisynthetic proteins and backbone cyclized polypeptides. The discovery of naturallyoccurring split-inteins has allowed the development of novel approaches for the selective modification of proteins both in vitro and in vivo. This review gives a general introduction to protein splicing with a focus on their role in expanding the applications of intein-based technologies in protein engineering and chemical biology.

Using backbone-cyclized Cys-rich polypeptides as molecular scaffolds to target protein-protein interactions.

The use of disulfide-rich backbone-cyclized polypeptides, as molecular scaffolds to design a new generation of bioimaging tools and drugs that are potent and specific, and thus might have fewer side effects than traditional small-molecule drugs, is gaining increasing interest among the scientific and in the pharmaceutical industries. Highly constrained macrocyclic polypeptides are exceptionally more stable to chemical, thermal and biological degradation and show better biological activity when compared with their linear counterparts. Many of these relatively new scaffolds have been also found to be highly tolerant to sequence variability, aside from the conserved residues forming the disulfide bonds, able to cross cellular membranes and modulate intracellular protein-protein interactions both in vitro and in vivo These properties make them ideal tools for many biotechnological applications. The present study provides an overview of the new developments on the use of several disulfide-rich backbone-cyclized polypeptides, including cyclotides, θ-defensins and sunflower trypsin inhibitor peptides, in the development of novel bioimaging reagents and therapeutic leads.

Two Blast-independent tools, CyPerl and CyExcel, for harvesting hundreds of novel cyclotides and analogues from plant genomes and protein databases.

Two high-throughput tools harvest hundreds of novel cyclotides and analogues in plants. Cyclotides are gene-encoded backbone-cyclized polypeptides displaying a diverse range of bioactivities associated with plant defense. However, genome-scale or database-scale evaluations of cyclotides have been rare so far. Here, a novel time-efficient Perl program, CyPerl, was developed for searching cyclotides from predicted ORFs of 34 available plant genomes and existing plant protein sequences from Genbank databases. CyPerl-isolated sequences were further analyzed by removing repeats, evaluating their cysteine-distributed regions (CDRs) and comparing with CyBase-collected cyclotides in a user-friendly Excel (Microsoft Office) template, CyExcel. After genome-screening, 186 ORFs containing 145 unique cyclotide analogues were identified by CyPerl and CyExcel from 30 plant genomes tested from 10 plant families. Phaseolus vulgaris and Zea mays were the richest two species containing cyclotide analogues in the plants tested. After screening protein databases, 266 unique cyclotides and analogues were identified from seven plant families. By merging with 288 unique CyBase-listed cyclotides, 510 unique cyclotides and analogues were obtained from 13 plant families. In total, seven novel plant families containing cyclotide analogues and 202 novel cyclotide analogues were identified in this study. This study has established two Blast-independent tools for screening cyclotides from plant genomes and protein databases, and has also significantly widened the plant distribution and sequence diversity of cyclotides and their analogues.

Recombinant expression of backbone‐cyclized polypeptides

Here we review the different biochemical approaches available for the expression of backbone-cyclized polypeptides, including peptides and proteins. These methods allow for the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Polypeptide circularization provides a valuable tool to study the effects of topology on protein stability and folding kinetics. Furthermore, having biosynthetic access to backbone-cyclized polypeptides makes the production of genetically encoded libraries of cyclic polypeptides possible. The production of such libraries, which was previously restricted to the domain of synthetic chemistry, now offers biologists access to highly diverse and stable molecular libraries that can be screened using high-throughput methods for the rapid selection of novel cyclic polypeptide sequences with new biological activities.

Biological Activities of Natural and Engineered Cyclotides, a Novel Molecular Scaffold for Peptide-Based Therapeutics

Cyclotides are a growing family of large plant-derived backbone-cyclized polypeptides (≈30 amino acids long) that share a disulfide-stabilized core characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Currently more than 100 sequences of different cyclotides have been characterized and the number is expected to increase dramatically in the coming years. Considering their stability, biological activities and ability to cross the cell membrane, cyclotides can be exploited to develop new peptide-based drugs with high potential for success. The cyclotide scaffold can be engineered or evolved using molecular evolution to inhibit protein-protein interactions implicated in cancer and other human diseases, or design new antimicrobial. The present review reports the biological diversity and therapeutic potential of natural and engineered cyclotides.

Biological Activities of Natural and Engineered Cyclotides, a Novel Molecular Scaffold for Peptide-Based Therapeutics

Cyclotides are a growing family of large plant-derived backbone-cyclized polypeptides (≉30 amino acids long) that share a disulfide-stabilized core characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Currently more than 100 sequences of different cyclotides have been characterized and the number is expected to increase dramatically in the coming years. Considering their stability, biological activities and ability to cross the cell membrane, cyclotides can be exploited to develop new peptide-based drugs with high potential for success. The cyclotide scaffold can be engineered or evolved using molecular evolution to inhibit protein-protein interactions implicated in cancer and other human diseases, or design new antimicrobials. The present review reports the biological diversity and therapeutic potential of natural and engineered cyclotides.

Cyclotides, a promising molecular scaffold for peptide-based therapeutics.

Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides (approximately 30 amino acids long) that share a disulfide-stabilized core (three disulfide bonds) characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds make them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Currently, more than 100 sequences of different cyclotides have been characterized, and the number is expected to increase dramatically in the coming years. Considering their stability and biological activities like anti-HIV, uterotonic, and insecticidal, and also their abilities to cross the cell membrane, cyclotides can be exploited to develop new stable peptide-based drugs. We have recently demonstrated the intriguing possibility of producing libraries of cyclotides inside living bacterial cells. This opens the possibility to generate large genetically encoded libraries of cyclotides that can then be screened inside the cell for selecting particular biological activities in a high-throughput fashion. The present minireview reports the efforts carried out toward the selection of cyclotide-based compounds with specific biological activities for drug design.

Expressed Protein Ligation: A New Tool for the Biosynthesis of Cyclic Polypeptides

The present paper reviews the use of expressed protein ligation for the biosynthesis of backbone cyclized polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes.