ABSTRACT
In the rapidly expanding field of peptide therapeutics, the short in vivo half-life of peptides represents a considerable limitation for drug action. D-peptides, consisting entirely of the dextrorotatory enantiomers of naturally occurring levorotatory amino acids (AAs), do not suffer from these shortcomings as they are intrinsically resistant to proteolytic degradation, resulting in a favourable pharmacokinetic profile. To experimentally identify D-peptide binders to interesting therapeutic targets, so-called mirror-image phage display is typically performed, whereby the target is synthesized in D-form and L-peptide binders are screened as in conventional phage display. This technique is extremely powerful, but it requires the synthesis of the target in D-form, which is challenging for large proteins. Here we present finDr, a novel web server for the computational identification and optimization of D-peptide ligands to any protein structure (https://findr.biologie.uni-freiburg.de/). finDr performs molecular docking to virtually screen a library of helical 12-mer peptides extracted from the RCSB Protein Data Bank (PDB) for their ability to bind to the target. In a separate, heuristic approach to search the chemical space of 12-mer peptides, finDr executes a customizable evolutionary algorithm (EA) for the de novo identification or optimization of D-peptide ligands. As a proof of principle, we demonstrate the validity of our approach to predict optimal binders to the pharmacologically relevant target phenol soluble modulin alpha 3 (PSMα3), a toxin of methicillin-resistant Staphylococcus aureus (MRSA). We validate the predictions using in vitro binding assays, supporting the success of this approach. Compared to conventional methods, finDr provides a low cost and easy-to-use alternative for the identification of D-peptide ligands against protein targets of choice without size limitation. We believe finDr will facilitate D-peptide discovery with implications in biotechnology and biomedicine.
ABSTRACT
Phytochromes are photoreceptors regulating growth and development in plants. Using the model plant Arabidopsis, we identified a novel signalling pathway downstream of the far-red light-sensing phytochrome, phyA, that depends on the highly conserved CCR4-NOT complex. CCR4-NOT is integral to RNA metabolism in yeast and animals, but its function in plants is largely unknown. NOT9B, an Arabidopsis homologue of human CNOT9, is a component of the CCR4-NOT complex, and acts as negative regulator of phyA-specific light signalling when bound to NOT1, the scaffold protein of the complex. Light-activated phyA interacts with and displaces NOT9B from NOT1, suggesting a potential mechanism for light signalling through CCR4-NOT. ARGONAUTE 1 and proteins involved in splicing associate with NOT9B and we show that NOT9B is required for specific phyA-dependent alternative splicing events. Furthermore, association with nuclear localised ARGONAUTE 1 raises the possibility that NOT9B and CCR4-NOT are involved in phyA-modulated gene expression.
Place a seedling on a windowsill, and soon you will notice the fragile stem bending towards the glass to soak in the sun and optimize its growth. Plants can 'sense' light thanks to specialized photoreceptor molecules: for instance, the phytochrome A is responsible for detecting weak and 'far-red' light from the very edge of the visible spectrum. Once the phytochrome has been activated, this message is relayed to the rest of the plant through an intricate process that requires other molecules. The CCR4-NOT protein complex is vital for all plants, animals and fungi, suggesting that it was already present in early life forms. Here, Schwenk et al. examine whether CCR4-NOT could have acquired a new role in plants to help them respond to far-red light. Scanning the genetic information of the plant model Arabidopsis thaliana revealed that the gene encoding the NOT9 subunit of CCR4-NOT had been duplicated in plants during evolution. NOT9B, the protein that the new copy codes for, has a docking site that can attach to both phytochrome A and CCR4-NOT. When NOT9B binds phytochrome A, it is released from the CCR4-NOT complex: this could trigger a cascade of reactions that ultimately changes how A. thaliana responds to far-red light. Plants that had not enough or too much NOT9B were respectively more or less responsive to that type of light, showing that the duplication of the gene coding for this subunit had helped plants respond to certain types of light. The findings by Schwenk et al. illustrate how existing structures can be repurposed during evolution to carry new roles. They also provide a deeper understanding of how plants optimize their growth, a useful piece of information in a world where most people rely on crops as their main source of nutrients.
