Invention of stimulus‐responsive peptide‐bond‐cleaving residue (Spr) and its application to chemical biology tools

Elucidation of biological functions of peptides and proteins is essential for understanding peptide/protein-related biological events and developing drugs. Caged peptides and proteins that release a parent active peptide/protein by photo-irradiation have successfully been employed to elucidate the functions. Whereas the usual caged peptide/protein enables conversion of an inactive form to an active form (OFF-to-ON conversion) by photo-induced deprotection, photo-triggered main chain cleavage is reported to be applicable to ON-to-OFF conversion. These peptides and proteins are photo-responsive; however, if peptides and proteins could respond to other stimuli such as disease-related environment or enzymes, their range of application should be widened. To convert the photo-responsive peptide/protein into other stimulus-responsive peptide/protein, quite laborious de novo design and synthesis of the stimulus-responsive unit are required. In this context, we designed a stimulus-responsive peptide-bond-cleaving residue (Spr) in which the stimuli available for the main chain cleavage vary according to the choice of protecting groups on the residue. In this review, design and synthesis of Spr are introduced, and challenges to apply Spr to other fields to enable, for example, functional control, localization control, delivery of cargos, labeling of a protein of interest in living cells, and identification of target proteins of bioactive ligands are discussed

Invention of stimulus‐responsive peptide‐bond‐cleaving residue (Spr) and its application to chemical biology tools

Elucidation of biological functions of peptides and proteins is essential for understanding peptide/protein-related biological events and developing drugs. Caged peptides and proteins that release a parent active peptide/protein by photo-irradiation have successfully been employed to elucidate the functions. Whereas the usual caged peptide/protein enables conversion of an inactive form to an active form (OFF-to-ON conversion) by photo-induced deprotection, photo-triggered main chain cleavage is reported to be applicable to ON-to-OFF conversion. These peptides and proteins are photo-responsive; however, if peptides and proteins could respond to other stimuli such as disease-related environment or enzymes, their range of application should be widened. To convert the photo-responsive peptide/protein into other stimulus-responsive peptide/protein, quite laborious de novo design and synthesis of the stimulus-responsive unit are required. In this context, we designed a stimulus-responsive peptide-bond-cleaving residue (Spr) in which the stimuli available for the main chain cleavage vary according to the choice of protecting groups on the residue. In this review, design and synthesis of Spr are introduced, and challenges to apply Spr to other fields to enable, for example, functional control, localization control, delivery of cargos, labeling of a protein of interest in living cells, and identification of target proteins of bioactive ligands are discussed

Time-series transcriptome of Brachypodium distachyon during bacterial flagellin-induced pattern-triggered immunity

Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of Brachypodium distachyon. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in B. distachyon. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction

N-Sulfanylethylanilide-based traceable linker for enrichment and selective labelling of target proteins

An N-sulfanylethylanilide-based traceable linker, developed to facilitate identification of target proteins of bioactive compounds, was introduced into an alkynylated target protein. Subsequent adsorption onto streptavidin beads allowed it to be treated with a cysteine-fluorophore conjugate in the presence of phosphate. This induced the N-S acyl transfer reaction of the N-sulfanylethylanilide unit. The subsequent native chemical ligation of the fluorophore resulted in cleavage of the linker for target elution and fluorescent labelling of the target, allowing it to be distinguished from non-target proteins

抱卵型カイアシ類Oithona oculata のバイオリアクターを用いた試験的培養

In aquaculture and ornamental industries, copepods are recognized as preferred live feeds for marine fish larvae over commonly used organisms such as Artemia and rotifers. Marine fish larvae fed with copepods show better survival and growth. Despite obvious advantages of copepods as live feed, their use is still limited owing to low productivity and cost-efficiency when mass cultured. Copepods can be divided into free-spawner and sac-spawner (egg-carrying) according to their spawning style. In cultivation of egg-carrying copepod, a simple nauplii collection/harvesting method with low labor has not been established, because females carry the egg-sac until hatching. Manual collection of nauplii using a siphon hose and mesh-filters is a common method in copepod cultivation, but automation is necessary to reduce labor costs if copepods are to be viably cultured at a commercial scale. Recently, we devised a zooplankton bioreactor for rapid separation of free-spawner copepod eggs from adults in a tank. The automation collects eggs on a mesh filter in a water flow system which can also function as water exchange device. In the present study, we apply this bioreactor to egg-carrying copepod, and report the results from cultivation at laboratory-scale for 45 days. Species of Oithona are good candidates for live feed because their body size and biochemical composition are suitable for many marine fish larvae which have small gapes. Oithona oculata is widely distributed and a typical dominant species in coastal waters, and was chosen as target species of egg-carrying copepod in present study. 150 adult individuals of O. oculata were placed into a culture chamber which were immersed in a reproduction tank containing 3 L seawater. The culture chamber to retain the copepods has a 100-μm nylon mesh placed 5 mm above the bottom, while allowing passage of nauplii. The copepods were fed daily with a sufficient amount of Thalassiosira weissflogii and Isochrysis galbana and incubated at 28°C for 45 days. Produced nauplii in the reproduction tank were collected daily using water flow of 1.5 L min-1. The species abundance in each development, ovigerous rate and collected number of nauplii were measured daily. In the semi-continuous culture, the collected nauplii were cultured in maturation vessels and returned to the production tank after their maturation, and 45 days cultivation (four generations) was achieved. Total abundance was gradually increased from day 4 to day 20 and reached at 500 inds. L-1. Ovigerous rate varied from 0 to 88% during the incubation period. The maximum number of collected nauplii was 380 nauplii L-1 at day 5. The results obtained in present study suggest that the bioreactor can also be applied to egg-carrying copepod by optimization of the mesh size and the water flow velocity

Design and synthesis of a hydrogen peroxide-responsive amino acid that induces peptide bond cleavage after exposure to hydrogen peroxide

Oxidative stress-responsive compounds are attracting significant attention in the field of medicinal chemistry and chemical biology. Here, we disclose the development of a hydrogen peroxide (H2O2)-responsive amino acid that induces peptide bond cleavage in the presence of H2O2 that closely relates to the oxidative stress. The H2O2-responsive amino acid possessing a boronate or boronic acid moiety was incorporated into a peptide using Fmoc-based solid-phase peptide synthesis or that with minor modification, respectively, and the peptide bond cleavage of the obtained peptide was successfully triggered by the addition of H2O2

Ribosome binding protein GCN1 regulates the cell cycle and cell proliferation and is essential for the embryonic development of mice.

Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation