Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

Naimuddin, M.; Kobayashi, Suzuko; Tsutsui, Chihiro; Machida, Masayuki; Nemoto, Naoto; Sakai, Takafumi; Kubo, Tai

doi:10.1186/1756-6606-4-2

Cited by 38 publications

(62 citation statements)

References 58 publications

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“…The success of TFPs in functional co-option has been attributed to their small size (~60-100 residues), lack of antigenicity, and extreme tolerance to temperature, various solvents, and most proteases [71]. These properties result from the highly disulfide-bonded, β-sheet scaffold that provides the namesake "three-finger" fold [72].…”

Section: Accepted Manuscriptmentioning

confidence: 98%

Co-option and evolution of non-olfactory proteinaceous pheromones in a terrestrial lungless salamander

Doty

Wilburn

Bowen

et al. 2016

Journal of Proteomics

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Gene co-option is a major force in the evolution of novel biological functions. In plethodontid salamanders, males deliver proteinaceous courtship pheromones to the female olfactory system or transdermally to the bloodstream. Molecular studies identified three families of highly duplicated, rapidly evolving pheromones (PRF, PMF, and SPF). Analyses for Plethodon salamanders revealed pheromone mixtures of primarily PRF and PMF. The current study demonstrates that in Desmognathus ocoee--a plesiomorphic species with transdermal delivery--SPF is the major pheromone component representing >30% of total protein. Chromatographic profiles of D. ocoee pheromones were consistent from May through October. LC/MS-MS analysis suggested uniform SPF isoform expression between individual male D. ocoee. A gene ancestry for SPF with the Three-Finger Protein superfamily was supported by intron-exon boundaries, but not by the disulfide bonding pattern. Further analysis of the pheromone mixture revealed paralogs to peptide hormones that contained mutations in receptor binding regions, such that these novel molecules may alter female physiology by acting as hormone agonists/antagonists. Cumulatively, gene co-option, duplication, and neofunctionalization have permitted recruitment of additional gene families for pheromone activity. Such independent co-option events may be playing a key role in salamander speciation by altering male traits that influence reproductive success.

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Section: Accepted Manuscriptmentioning

confidence: 98%

Co-option and evolution of non-olfactory proteinaceous pheromones in a terrestrial lungless salamander

Doty

Wilburn

Bowen

et al. 2016

Journal of Proteomics

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“…Recently, we have developed an in vitro display system, termed cDNA display, which is an improved system of mRNA display; some peptides were successfully screened via this method (42)(43)(44)(45)(46). Because a peptide is displayed on a cDNA in cDNA display (instead of being displayed on mRNA, as with mRNA display), peptide-cDNA fusion libraries resist degradation by ribonucleases and can be used under severe selection conditions, such as cell culture medium.…”

mentioning

confidence: 99%

In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display

Ueno

Yoshida

Mondal

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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G protein-coupled receptors (GPCRs) are major drug targets, and their ligands are currently being explored and developed by many pharmaceutical companies and independent researchers. Class A (rhodopsin-like) GPCRs compose a predominant GPCR family; therefore, class A GPCR ligands are in demand. Growth hormone secretagogue receptor (GHS-R) is a class A GPCR that stimulates food intake by binding to its peptide ligand, ghrelin. Therefore, antagonists of GHS-R are expected to exert antiobesity function. In this article, we describe the use of cDNA display to screen for successfully and identify an antagonistic peptide of GHS-R. The antagonistic peptide inhibited the ghrelin-induced increase in intracellular Ca 2þ in vitro (IC 50 ¼ approximately 10 μM) and repressed the contraction of isolated animal stomach in response to ghrelin. Furthermore, peripheral administration of the peptide inhibited the food intake of mice. This work provides new insight into the development of antiobesity drugs and describes a method for the discovery of unique peptide ligands for class A GPCRs.aptamer | in vitro display | peptide drug | ligand screening | cell-based selection

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“…This approach was recently applied using cDNA display on a three-finger scaffold, in order to produce novel molecules such as interleukin-6 receptor ligands 21 and serine protease inhibitors 22 . Nevertheless, in order to obtain smaller but functionally rich libraries, rational protein engineering was developed by exploiting information on protein sequence, Figure 8.…”

Section: Discussionmentioning

confidence: 99%

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

Blanchet

Stura

Mourier

et al. 2018

Toxicon

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Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α 1A -adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α 1 and α 2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.Evolutionary processes of venomous animals have selected enzymes and disulfide-rich peptides in their venoms to improve their ability to subdue their prey and defend against predators. These compounds act predominantly on few, well characterized and physiologically-relevant molecular targets of the envenomed animals 1 . Recruited by convergent evolution, toxins impact mainly the haemostatic, nervous and cardiovascular systems 2 . Some toxins present in venoms, in spite of their debilitating effects, have become live-saving drugs [3][4][5] . To exert their biological activities, toxins interact mainly with ion channels, coagulation factors, nicotinic receptors, cell membrane or enzymes but only rarely with the most important class of physiological targets, i.e, the G protein-coupled receptors (GPCRs) 6 . Compared to numerous toxins that act on voltage-gated or ligand-gated ion channels, only a few toxins that interact with GPCRs have been identified. These toxins have been isolated mainly from cone snails venoms such as conopressin interacting with vasopressin receptor 7 , contulakin-G which binds to neurotensin receptor 8 , ρ-conotoxin TIA specific of the α 1A -adrenoceptor 9 and the τ-conotoxin CnVA that interacts with the somatostatin sst3 receptor 10 . The black widow spider and the gila monster provide further examples of GPCRs interacting toxins, namely, α-latrotoxin interacting with the latrophilin receptor 11 and exenatide, exploited as an anti-diabetic drug 12 , that targets the GLP-1 receptor, respectively. In addition, mamba venoms contain aminergic toxins that recognize various bioaminergic receptors [13][14][15][16][17][18][19] . Aminergic toxins belong to the three-finger fold toxin (3FT) superfamily, a structural fold known to support a large diversity of...

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Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

Cited by 38 publications

References 58 publications

Co-option and evolution of non-olfactory proteinaceous pheromones in a terrestrial lungless salamander

Co-option and evolution of non-olfactory proteinaceous pheromones in a terrestrial lungless salamander

In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

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